Gut microbiota alteration with growth performance, histopathological lesions, and immune responses in Salmonella Typhimurium-infected weaned piglets.

Salmonella Typhimurium can cause gastroenteritis in weaned piglets, which are particularly vulnerable to dietary changes and dysfunction of their immature organs. The colonization of S. Typhimurium could disrupt the gut microbiota and increase susceptibility to the bacterium. This study aimed to investigate the alterations of gut microbiota in S. Typhimurium-infected weaned piglets. Ten 49-day-old pigs were divided into two groups: S. Typhimurium-inoculated (ST, n = 6) and negative control (NC, n = 4) groups. The body weight and S. Typhimurium fecal shedding were monitored for 14 days after S. Typhimurium inoculation (dpi). The intestinal tissues were collected at 14 dpi; histopathological lesions and cytokine gene expression were evaluated. The gut microbiome composition and short-chain fatty acid concentrations were analyzed in fecal samples collected at 14 dpi. The average daily gain and gut microbiota alpha diversity in ST group tended to be lower than NC group at 14 dpi. Linear discriminant analysis effect size results showed a significant increase in the abundance of two genera and five species, while a significant decrease was observed in the five genera and nine species within the gut microbiota of ST group. Among the significantly less abundant bacteria in the ST group, Lachnospira eligens and Anaerobium acetethylicum produce acetate and butyrate, and may be considered as key S. Typhimurium infection-preventing bacteria. The overall results provide invaluable information about changes in the gut microbiota of S. Typhimurium-infected weaned piglets, which can be used to develop alternative measures to antibiotics and prevent ST bacterial infection.

Raw potato starch diet supplement in weaned pigs could reduce Salmonella Typhimurium infection by altering microbiome composition and improving immune status.

Backgorund: Salmonella enterica serovar Typhimurium (ST) is one of the causative agents of gastroenteritis in pigs. Pigs fed a diet supplemented with raw potato starch (RPS) have improved gut health by the alteration of the microbiota composition and production of short-chain fatty acids (SCFAs). This study aimed to evaluate the effects of RPS supplementation in reducing infection severity and fecal shedding in ST-infected pigs. Methods: The weaned experimental pigs were divided into two groups: CON (n = 6) fed a corn/soybean-based diet and TRT (n = 6) supplemented with 5% RPS. After 21 d, the pigs were inoculated with ST, and their body weight, clinical signs, and fecal shedding of ST were monitored for 14 d. At 14 d post-inoculation (dpi), the jejunum, cecum, ileum, and colon tissues were collected from euthanized pigs, and histopathological lesions and cytokine gene expression were compared. Additionally, blood samples at 2 dpi were analyzed for gene ontology enrichment. Moreover, the gutmicrobiome was analyzed using 16S rRNA metagenomic sequencing, and the SCFA concentration was measured using gas chromatography. Results: The average daily weight gain was significantly higher in TRT than in CON during the ST infection period; however, histopathological lesion scores were significantly lower in TRT than in CON. The relative abundance of nine genera of butyrate- and acetate-producing bacteria significantly increased in TRT compared with that of only two acetate-producing bacteria in CON. Among the genes involved in the immune response, IL-18 expression level was significantly lower in the jejunum and colon in TRT than in CON. Furthermore, Reg3γ expression was significantly different in the cecum and colon of both groups. Conclusion: The diet supplemented with RPS in weaned pigs could result in predominance of butyrate- and acetate-producing bacteria, reducing the severity of ST infection by improving the immune status.

Multi-organ metastases of primary chondrosarcoma of the mammary gland of a spayed Toy Poodle.

We report the clinical and histopathological findings associated with multi-organ metastases of primary mammary chondrosarcoma in a 12-year-old spayed female Toy Poodle. At post-mortem examination, multifocal, sharply demarcated, grey-white to bright brown, round nodules of variable size were randomly distributed in the lungs, myocardium, liver, pancreas, spleen, intestinal tract and kidneys. Histologically, immature cartilage structures and primitive mesenchymal cells were seen in these organs. Neoplastic cells located in the cartilaginous basophilic extracellular matrix had cytoplasmic vacuolation and round vesicular nuclei and were stained with Safranin O and Alcian blue. To the best of our knowledge, this is the first report of a multi-organ metastatic chondrosarcoma that originated in the mammary gland of a dog.

Johne's disease in two Korean black goats (Capra hircus coreanae) with clinical and subclinical infection: A case report.

Johne's disease (JD) is a chronic enteric infection in ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). JD infection is more difficult to diagnose in goats than cattle because MAP can insidiously affect small ruminants. Few reports have used pathological and molecular diagnosis for cases in Korean black goats. Here, we present our results from two MAP-infected goats. Case 1 was categorized as clinically significant (stage IV), with severe clinical signs and a high antibody titer (S/P ratio, 158.9%). Case 2 was in the subclinical stage (stage II); however, the goat suddenly died without any clinical signs (S/P ratio, 70.9%). DNA from the organ tissues and feces from Case 1 showed a strong positive PCR result for MAP, whereas Case 2 only exhibited a very weak reaction in the fecal sample. Moreover, fecal DNA from both cases was genotyped as C-type MAP using the PCR-REA method. Gastrointestinal organ tissues (jejunum, ileum, colon, and mesenteric lymph nodes) from Case 1 showed moderate-to-severe lesions, and acid-fast bacilli were observed. In contrast, Case 2 showed intact-to-mild pathological lesions, and acid-fast bacilli were detected in the colon, mesenteric lymph nodes, and liver. In addition, Case 2 was co-infected with Corynebacterium pseudotuberculosis, which caused caseous lymphadenitis. This case study provides valuable information regarding the pathological and molecular characteristics of JD-infected Korean black goats. The results highlighted the differences in pathological lesions between clinically and subclinically infected goats, which help veterinarians to develop better strategies to control MAP in goat farms.

Blood parameters and pathological lesions in pigs experimentally infected with Vietnam's first isolated African swine fever virus.

African swine fever virus (ASFV) is a notable virus and one of the most serious global threats to the pig industry. Improving awareness about host-virus interactions could facilitate the understanding of the disease pathogenesis. Therefore, we investigated changes in blood parameters, viral loads, and pathological changes in ASFV-inoculated pigs according to the time of death after the onset of viremia. For the analyses, the ASFV-infected pigs (n = 10) were divided into two groups (five pigs/group) according to their time of death after the onset of viremia. The blood cell count dynamics and serum biochemistry profiles were similar between the groups; however, viral load distribution was different. A comparison of the histopathological changes and immunohistochemistry results between the two groups indicated that the lymphoid system, particularly the spleen, was more damaged in the early stage of the disease than in the last stage. Additionally, the virus-induced lesions in other organs (liver and kidney) were more severe in the late stage than in the early stage. Our findings provide invaluable information on the characteristics of blood parameters and pathological lesions in pigs infected with the Asia-epidemic ASFV strain and the course of ASF, targeting internal organs in pigs. Overall, this study characterizes the host-pathogen interaction in ASFV infection, offering insight for the establishment of ASF control strategies.

Effect of feeding raw potato starch on the composition dynamics of the piglet intestinal microbiome.

OBJECTIVE: Raw potato starch (RPS) is resistant to digestion, escapes absorption, and is metabolized by intestinal microflora in the large intestine and acts as their energy source. In this study, we compared the effect of different concentrations of RPS on the intestinal bacterial community of weaned piglets. METHODS: Male weaned piglets (25-days-old, 7.03±0.49 kg) were either fed a corn/soybean-based control diet (CON, n = 6) or two treatment diets supplemented with 5% RPS (RPS5, n = 4) or 10% RPS (RPS10, n = 4) for 20 days and their fecal samples were collected. The day 0 and 20 samples were analyzed using a 16S rRNA gene sequencing technology, followed by total genomic DNA extraction, library construction, and high-throughput sequencing. After statistical analysis, five phyla and 45 genera accounting for over 0.5% of the reads in any of the three groups were further analyzed. Furthermore, short-chain fatty acids (SCFAs) in the day 20 fecal samples were analyzed using gas chromatography. RESULTS: Significant changes were not observed in the bacterial composition at the phylum level even after 20 d post feeding (dpf); however, the abundance of Intestinimonas and Barnesiella decreased in both RPS treatment groups compared to the CON group. Consumption of 5% RPS increased the abundance of Roseburia (p<0.05) and decreased the abundance of Clostridium (p<0.01) and Mediterraneibacter (p< 0.05). In contrast, consumption of 10% RPS increased the abundance of Olsenella (p<0.05) and decreased the abundance of Campylobacter (p<0.05), Kineothrix (p<0.05), Paraprevotella (p<0.05), and Vallitalea (p<0.05). Additionally, acetate (p<0.01), butyrate (p<0.05), valerate (p = 0.01), and total SCFAs (p = 0.01) were upregulated in the RPS5 treatment group. CONCLUSION: Feeding 5% RPS altered bacterial community composition and promoted gut health in weaned piglets. Thus, resistant starch as a feed additive may prevent diarrhea in piglets during weaning.

Erratum to: Evaluation of adenosine triphosphate testing for on-farm cleanliness monitoring compared to microbiological testing in an empty pig farrowing unit.

[This corrects the article DOI: 10.5187/jast.2020.62.5.682.].

Age-dependent immune response in pigs against foot-and-mouth disease virus in vitro.

Foot-and-mouth disease, one of the most contagious diseases in cloven-hoofed animals, causes significant economic losses. The pathogenesis of foot-and-mouth disease virus (FMDV) infection is known to differ with age of the animals. In this study, we aimed to reveal the difference in immunological response in the initial stage of FMDV infection between piglets and adult pigs. Peripheral blood mononuclear cells (PBMCs) were isolated from 3 piglets (8 weeks old) and 3 pigs (35 weeks old) that were not vaccinated against FMDV. O-type FMDV (2 × 102 median tissue culture infectious dose) was inoculated into porcine PBMCs and the cells were incubated at 37.0°C under 5% CO2 for various time periods (0, 1, 3, 6, 12, 24, and 48 h). The total RNA was obtained from the FMDV-inoculated PBMCs after each time point, and the virus titer was investigated in these RNA samples. Furthermore, dynamics of mRNA expression of the six tested cytokines (interferon [IFN]-α, IFN-γ, interleukin [IL]-6, IL-8, IL-10, and tumor necrosis factor [TNF]-α) in FMDV-inoculated porcine PBMCs were evaluated by time-series analysis to determine the differences, if any, based on the age of the pigs. The PBMCs of piglets contained the highest quantity of FMDV mRNA at 6 hours post-inoculation (hpi), and the PBMCs of pigs had the highest quantity of FMDV mRNA at 3 hpi. The mean cycle threshold-value in the PBMCs steadily decreased after the peak time point in the piglets and pigs (6 and 3 hpi, respectively). The dynamics of mRNA expression of all cytokines except TNF-α showed age-dependent differences in FMDV-inoculated PBMCs. The mRNA expression of most cytokines was more pronounced in the piglets than in the pigs, implying that the immune response against FMDV showed an age-dependent difference in pigs. In conclusion, within 48 hpi, the 8-week-old piglets responded more rapidly and were more sensitive to FMDV infection than the 35-week-old pigs, which could be associated with the difference in the pathogenesis of FMDV infection among the pigs. These results provide valuable insights into the mechanisms underlying the age-dependent differences in immune response in pigs against FMDV infection.

Dynamic Variations in Infrared Skin Temperature of Weaned Pigs Experimentally Inoculated with the African Swine Fever Virus: A Pilot Study.

African swine fever (ASF) is a devastating viral disease in pigs and is therefore economically important for the swine industry. ASF is characterized by a short incubation period and immediate death, making the early identification of ASF-infected pigs essential. This pilot-scale study evaluates whether the infrared thermography (IRT) technique can be used as a diagnostic tool to detect changes in skin temperature (Tsk) during the early stages of disease development in experimentally ASF-infected pigs. Clinical symptoms and rectal temperatures (Tcore) were recorded daily, and IRT readings during the experimental ASF infection were analyzed. All infected pigs died at 5-8 days post inoculation (dpi), and the incubation period was approximately 4 dpi. The average Tcore increased from 0 dpi (38.9 ± 0.3 °C) to 7 dpi (41.0 ± 0.5 °C) and decreased by 8 dpi (39.8 ± 0 °C). The maximum Tsk of ASF-infected pigs increased from 2 (35.0 °C) to 3 dpi (38.5 °C). The mean maximum Tsk observed from three regions on the skin (ear, inguinal, and neck) significantly increased from 2 to 3 dpi. This study presents a non-contact method for the early detection of ASF in infected pigs using thermal imaging at 3 days after ASF infection.

Incidence of inadvertent intercostal or epidural spread during thoracic sympathetic ganglion block.

BACKGROUND: Sympathetic blocks (SBs) have been used widely to relieve the symptoms of sympathetically maintained pain (SMP). The thoracic sympathetic ganglion is not separated from somatic nerves by muscles and connective tissue. The upper thoracic ganglion runs along the posterior surface of the vertebral column in close proximity to the adjacent epidural region. This anatomical difference leads to frequent epidural and intercostal spread in cases of thoracic SBs. The purpose of this study was to investigate the incidence of inadvertent intercostal and epidural injections during thoracic SBs. METHODS: Twenty-two patients who were suffering from complex regional pain syndrome or lymphedema after breast cancer surgery were managed with two or three times of thoracic SBs. Therefore, injections of 63 thoracic SBs from 22 patients were enrolled in this study. An investigator who did not attend the procedure evaluated the occurrence of intercostal or epidural spread using anteroposterior fluoroscopic images. RESULTS: The overall incidence of inadvertent intercostal or epidural spread of contrast was 47.5%. Among the inadvertent injections, intercostal spread (34.9%) was more frequent than epidural spread (12.6%). Only 52.5% of the thoracic SBs demonstrated successful contrast spread without any inadvertent spread. The mean difference in skin temperature between the blocked and unblocked sides was 2.5 ± 1.8ºC. Fifty-nine (93.6%) injections demonstrated more than 1.5ºC difference. CONCLUSIONS: Thoracic SBs showed a high incidence (47.5%) of inadvertent epidural or intercostal injection. Thus, special attention is required for the diagnosis of SMP or the injection of any neurolytic agent around sympathetic ganglion.

Evaluation of adenosine triphosphate testing for on-farm cleanliness monitoring compared to microbiological testing in an empty pig farrowing unit.

Careful cleaning and disinfection of pigpens is essential to prevent disease spread and avoid the resultant economic losses. Hygiene in pigpens is generally evaluated by visual monitoring supplemented with bacteriological monitoring, which includes counting the total aerobic bacteria (TAB) and/or fecal indicator bacteria (FIB). However, these methods present drawbacks such as time and labor requirements. As adenosine triphosphate (ATP) is ubiquitous in all living organisms including microorganisms, this study aimed to directly compare the results of microbial assessment and ATP quantification, and to suggest possible detailed application methods of the ATP test for hygiene evaluation in pigpens of a farrowing unit. Before and after standard cleaning procedures, samples were collected from the floor corner, floor center, and feeding trough of four pigpens at different time points. No FIB were detected and both the TAB and ATP levels were significantly decreased in the floor center area after cleaning. FIB were continuously detected after cleaning and disinfection of the floor corners, and there was no significant ATP level reduction. The feeding trough did not show any significant difference in these values before and after cleaning, indicating insufficient cleaning of this area. The levels of TAB and ATP after cleaning were significantly correlated and the average ATP value was significantly lower in the absence of FIB than in their presence. In the absence of standard references, a more thorough hygiene management could be achieved evenly by supplementing cleaning or disinfection based on the lowest ATP results obtained at the cleanest test site, which in the present study was the floor center. Overall, these results indicate that the on-farm ATP test can be used to determine the cleanliness status, in addition to visual inspection, as an alternative to laboratory culture-based testing for the presence of microorganisms.

Prevalence of poultry red mite (Dermanyssus gallinae) in Korean layer farms and the presence of avian pathogens in the mite.

The poultry red mite, Dermanyssus gallinae, is a blood-feeding parasite of layer hens and a potential vector of several avian infectious agents. High infestation with D. gallinae in layer farm buildings could result in economic losses, and the mites may act as a reservoir of avian pathogens within farms. This study aimed to assess the prevalence of D. gallinae in layer farm buildings in Korea and to investigate avian pathogens in the collected mites. The mite samples were collected from 36 Korean layer farm buildings on 21 farms nationwide. Information obtained from each farm building included the flock size, flock age, methods for controlling D. gallinae, and cleaning status. Association between these variables and the population density of D. gallinae was analyzed. Additionally, the presence of 10 avian pathogens was assessed using DNA samples from mites collected in 16 farm buildings. The prevalence of D. gallinae was 75% at the farm building level (90.5% at the farm level). Repetitive cleaning procedures for each building were significantly related with the mite infestation level, and the most influential factor for determining the mite population in the layer farm buildings. In the 16 DNA samples, we detected avian pathogenic Escherichia coli (n = 6), wild-type fowlpox virus (n = 3), wild-type Marek's disease virus (n = 2), chicken anemia virus (n = 1), and fowl adenovirus (n = 1). These findings suggest that repetitive cleaning procedures for the layer farm buildings could decrease the numbers of D. gallinae which may transmit avian pathogens within the farm.

High prevalence of blaCTX-M group genes in Aeromonas dhakensis isolated from aquaculture fish species in South Korea.

The prevalence of resistant genes against ß-lactams in 119 Aeromonas strains was determined. A large number (99.2%) of the present fish strains were resistant to one or more ß- lactams including ceftiofur, amoxicillin-clavulanic acid, ampicillin, piperacillin and cefpodoxime. Among antibiotic resistance phenotypes, the simultaneous resistance to all ß-lactams occurred in 25.2% (n=30) of all strains, which consisted of 18 strains of A. dhakensis, 8 strains of A. caviae, 2 strains of A. hydrophila and only one strain of A. veronii. For exploring genetic background of the antibiotic resistances, multiple PCR assays were subjected to detect ß-lactamase-encoding genes, bla(TEM), bla(OXA-B) and bla(CTX-M). In the results, the bla(TEM-1) gene was harbored in all strains, whereas only 3 strains harbored bla(OXA) gene. In the case of bla(CTX-M) gene, the gene was detected in 21.0% (25 out of 119) of all strains, which countered with 80% (20 out of 25) of A. dhakensis, 8% (2 out of 25) of A. caviae and 12% (3 out of 25) of A. hydrophila. In addition, most of the bla(CTX-M) positive strains showed simultaneous resistance to all ß-lactams (18 out of 30 strains). In sequence analysis for bla(CTX-M) genes detected, they were CTX-M group 1-encoding genes including bla(CTX-M-33) from 3 eel strains of A. dhakensis. Therefore, A. dhakensis obtained from cultured fish could represent a reservoir for spreading genes encoding CTX-M group 1 enzymes and hence should be carefully monitored, especially for its potential risk to public health.

Severe sepsis due to Aeromonas aquariorum in a patient with liver cirrhosis.

Thus far, Aeromonas aquariorum infection has been unrecorded in Korea. Herein, we report a fatal case of A. aquariorum infection in a 77-year-old male patient with liver cirrhosis. The bacterium isolated from a blood culture was initially mistaken as Aeromonas hydrophila using the Vitek2 identification system. In spite of intravenous ceftriaxone therapy, the patient was exacerbated by multiple organ dysfunction. By 4 days after admission, there was no hope for treatment or remission of symptoms and the patient was discharged. In the detailed microbiological investigations, the bacterium was identified as A. aquariorum harboring the act and alt genes, which encode cytotoxic and cytotonic enterotoxins.

Molecular characterization of Aeromonas species isolated from farmed eels (Anguilla japonica).

Seventy Aeromonas strains were identified by phylogenetic analysis using housekeeping genes (gyrB and rpoD) in order to investigate etiological agents for aeromoniasis in farmed eels (Anguilla japonica). The phylogenetic analysis showed that Aeromonas aquariorum (n=22, 31.4%) was the predominant species among the investigated eel strains, followed by Aeromonas caviae (n=16, 22.9%), A. veronii (n=13, 18.6%), A. hydrophila (n=12, 17.1%), A. jandaei (n=4, 5.7%), A. media (n=2, 2.9%), and A. trota (n=1, 1.4%). The potential virulence of the present strains was estimated by performing PCR assays using the following seven virulence genes: cytotoxic enterotoxin (act), two cytotonic enterotoxins (alt and ast), glycerophospholipid:cholesterol acyltransferase (gcaT), DNase (exu), lipase (lip), and flagellin (fla). The detection rates of act, alt, ast, gcaT, exu, lip, and fla among all 70 strains were 91.4%, 55.7%, 27.1%, 97.1%, 95.7%, 100%, and 98.6%, respectively. In genotyping of enterotoxin genes, act(+)/alt(+)/ast(+), act(+)/alt(+)/ast(-), and act(+)/alt(-)/ast(-) genotypes were prevalent in A. hydrophila (8/12 strains), A. aquariorum (13/22 strains), and A. caviae (14/16 strains), respectively, suggesting a high heterogeneity among Aeromonas species. In this study, A. aquariorum, which has been an unrecorded species in Korea, can be an etiological agent for aeromoniasis of eel.

Bone density measurement in interdental areas with simulated placement of orthodontic miniscrew implants.

INTRODUCTION: The purposes of this study were to determine bone density at various orthodontic implant sites and compare them according to depth and area (anterior and posterior, buccal and lingual, and maxilla and mandible). METHODS: Maxillofacial computed tomography scan data were obtained from 30 adults with normal occlusion. Bone density was recorded in Hounsfield units with simulated placement of miniscrews with the V-Implant program (CyberMed, Seoul, Korea). Bone density was measured to a depth of 6 mm at 1-mm intervals in 60 interdental areas (30 in the maxilla, 30 in the mandible), and mean bone density was calculated at each site. RESULTS: Bone density tended to decrease with increasing depth, particularly in the posterior area. Mean bone density showed a progressive increase from posterior to anterior except for the mandibular buccal side, which had no significant differences. A comparison of the mean bone densities between the buccal and lingual sides in the mandible showed that the lingual side had higher values in the anterior area and vice versa in the posterior area. On the other hand, there were no distinct differences between the buccal and lingual sides in the maxilla. A comparison of the mean bone densities between the maxilla and the mandible showed higher values in the mandible, and these differences were more significant on the buccal side of the posterior. CONCLUSIONS: The differences in bone densities according to depth and area should be considered when selecting and placing miniscrew implants for orthodontic anchorage.

Synchrotron radiation imaging of internal structures in live animals.

Ionizing radiation has long been used in medicine since the discovery of X-rays. Diagnostic imaging using synchrotron radiation has been under investigation since Rubenstein et al. reported dual-energy iodine-K-edge subtraction coronary angiography. Recently, computed tomography (CT) and magnetic resonance imaging (MRI) have provided better quality results than conventional radiology, providing important information on human internal structures. However, such techniques are unable to detect fine micron sized structures for the early diagnosis of tumors, vascular diseases and other medical objectives. Third generation synchrotron X-rays are well known for their superiority in coherence and energy tunability with respect to conventional X-rays. Consequently, new contrast mechanisms with a superior spatial resolution are becoming available. Here we present the extremely fine details of live animal internal structures using unmonochromatized synchrotron X-rays (white beam) and a simple detector system. Natural movements of the internal organs are also shown. The results indicate that this imaging technique can be applied to investigating microstructures and evaluating the function of the internal organs. Furthermore, this imaging system may be applied to humans as the next tool beyond CT and MRI.