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.

The Cxadr-Adam10 complex plays pivotal roles in tight junction integrity and early trophoblast development in mice.

Understanding preimplantation embryo development has important implications for assisted reproductive technologies (ARTs) after the introduction of in vitro fertilisation and embryo transfer because most embryonic losses occur during pre/peri-implantation. Recent studies have shown that tight junctions (TJs) are important components for embryos to develop to the blastocyst stage. However, their biological function after cavitation has not been extensively studied. We examined TJ assembly focusing on coxsackievirus and adenovirus receptor (Cxadr) and A disintegrin and metalloproteinase 10 (Adam10) using siRNA and/or an Adam10-specific inhibitor (GI254023X). TJ-associated genes, including occludin and tight junction protein 1 (Tjp1), were downregulated in the Cxadr knockdown (KD) embryos but were unaltered in Adam10 KD embryos. However, Adam10 KD or chemical inhibition affected subcellular localisation of Adam10, Cxadr, and Tjp1, leading to disrupted TJ assembly. Furthermore, Cxadr KD or GI254023X-treated blastocysts showed a relatively smaller outgrowth area and aberrant expression of transcription factor AP-2γ, a trophoblast-specific marker in the in vitro embryo outgrowth assay. In summary, we demonstrated that the Cxadr-Adam10 complex might moderate TJ integrity/stability and play pivotal roles during early embryonic development. Collectively, understanding the establishment of the TJ complex and its integrity will provide insight into translational research for predicting and selecting developmental competency for ART.

Characterisation of the bacterial community in the gastrointestinal tracts of elk (Cervus canadensis).

The resident bacteria of the gastrointestinal tract (GIT) and the behaviour of these microbes have been poorly characterised in elk as compared to other ruminant animal species such as sheep and cattle. In addition, most microbial community studies of deer gut have focused on rumen or faeces, while other parts of the GIT such as the small and large intestine have received little attention. To address this issue, the present study investigated the diversity of the GIT bacterial community in elk (Cervus canadensis) by 16S rRNA pyrosequencing analysis. Eight distinct GIT segments including the stomach (rumen, omasum, and abomasum), small intestine (duodenum and jejunum), and large intestine (cecum, colon, and rectum) obtained from four elks were examined. We found that bacterial richness and diversity were higher in the stomach and large intestine than in the small intestine (P < 0.05). A total of 733 genera belonging to 26 phyla were distributed throughout elk GITs, with Firmicutes, Bacteroidetes, and Proteobacteria identified as the predominant phyla. In addition, there was spatial heterogeneity in the composition, diversity, and species abundance of microbiota in the GIT (P < 0.0001). To the best of our knowledge, this is the first study to characterise bacterial communities from eight GIT regions of elk by 16S rRNA pyrosequencing.

Genotypic Classification of Staphylococcus aureus and Bacillus cereus from Korean Slaughterhouses Using Semiautomated Repetitive Sequence-Based Polymerase Chain Reaction.

A repetitive sequence-based polymerase chain reaction (rep-PCR) technique utilizing a semiautomated system, namely DiversiLab, was applied to determine the genotypes of Staphylococcus aureus and Bacillus cereus obtained from slaughterhouses. Twenty-four S. aureus and 16 B. cereus isolates from pigs and Hanwoo cattle from three slaughterhouses were used to create a DNA fingerprint library with the system software. Scatterplots demonstrated that rep-PCR groupings of S. aureus isolates were in good agreement with their origins. Specifically, linked rep-PCR profiles were observed for S. aureus isolates recovered from the same slaughterhouse, and higher genetic similarities were found among strains isolated from adjacent regions. All S. aureus isolates except one (ID: A-Hanwoo-9) from slaughterhouse "A" clustered with the three S. aureus reference strains, Korea Culture Center of Microorganisms (KCCM) 41291, KCCM 12214, and Culture Collection of Antimicrobial Resistant Microbes (CCARM) 3A007 (similarity values >95%). Moreover, most isolates obtained from slaughterhouse "B" clustered with S. aureus KCCM 11335 and KCCM 41331, and two isolates from slaughterhouse "C" clustered with CCARM 0027. Therefore, for this species, genotypic characteristics of regional isolates can be used to track the pathway of contamination. In contrast, B. cereus isolates showed high genetic diversity and could not be clustered with any specific group. Collectively, this system is useful for analyzing genetic diversity and is a rapid and reproducible typing method; however, there is a need to develop rep-PCR libraries for its use as a rapid identification method.

Detection and Identification of Sarcocystis cruzi (Protozoa: Apicomplexa) by Molecular and Ultrastructural Studies in Naturally Infected Korean Cattle (Bos taurus coreanae) from Daejeon, Korea.

To survey the prevalence of Sarcocystis infections, 210 heart samples were collected from Korean native cattle (Bos taurus coreanae) at an abattoir in Daejeon Metropolitan City, Republic of Korea. Sarcocysts were detected form 31 specimens (14.8%) and identified as Sarcocystis cruzi via transmission electron microscopy. The wall of S. cruzi has flattened protrusions that did not contain fibrils or microfilaments. The protrusions arose irregularly from the base, contained a fine granular substance, lacked internal microfilaments, and measured approximately 0.21-1.25 µm in length and 0.05-0.07 µm in width. Sequence analysis revealed 99.5% homology to S. cruzi. This is the first report on the prevalence of S. cruzi in native cattle from the Republic of Korea.

Influences of somatic donor cell sex on in vitro and in vivo embryo development following somatic cell nuclear transfer in pigs.

OBJECTIVE: The present study investigates pre- and post-implantation developmental competence of nuclear-transferred porcine embryos derived from male and female fetal fibroblasts. METHODS: Male and female fetal fibroblasts were transferred to in vitro-matured enucleated oocytes and in vitro and in vivo developmental competence of reconstructed embryos was investigated. And, a total of 6,789 female fibroblast nuclear-transferred embryos were surgically transferred into 41 surrogate gilts and 4,746 male fibroblast nuclear-transferred embryos were surgically transferred into 25 surrogate gilts. RESULTS: The competence to develop into blastocysts was not significantly different between the sexes. The mean cell number of female and male cloned blastocysts obtained by in vivo culture (143.8±10.5 to 159.2±14.8) was higher than that of in vitro culture of somatic cell nuclear transfer (SCNT) groups (31.4±8.3 to 33.4±11.1). After embryo transfer, 5 pregnant gilts from each treatment delivered 15 female and 22 male piglets. The average birth weight of the cloned piglets, gestation length, and the postnatal survival rates were not significantly different (p<0.05) between sexes. CONCLUSION: The present study found that the sex difference of the nuclear donor does not affect the developmental rate of porcine SCNT embryos. Furthermore, postnatal survivability of the cloned piglets was not affected by the sex of the donor cell.

Differential gene-expression profiles from canine cumulus cells of ovulated versus in vitro-matured oocytes.

We compared the nuclear maturation status and gene-expression profiles of canine cumulus cells (CCs) derived from cumulus-oocyte complexes (COCs) that were spontaneously ovulated versus those that were matured in vitro. Cumulus-oocyte complexes were retrieved from uteri by surgical flushing (after spontaneous ovulation) or by ovariectomy follicle aspiration and in vitro maturation. The objective of Experiment 1 was to investigate the nuclear maturation status of in vivo- versus in vitro-matured oocytes. The objective of Experiment 2 was to compare gene-expression profiles of CCs derived from in vivo- versus in vitro-matured COCs. Genes analysed are related to cell maturation, development and apoptosis, including GDF9, MAPK1, PTX3, CX43, Bcl2 and BAX; mRNA expression for all of these genes, except for GDF9, differed (P<0.05) between in vivo- and in vitro-matured CCs. In conclusion, we found that gene-expression profiles are related to the quality of CCs and therefore posit that monitoring gene expression could be a useful strategy to guide attempts to improve in vitro culture systems.

Identification of bovine viral diarrhea virus infection in Saanen goats in the Republic of Korea.

Bovine viral diarrhea virus (BVDV) is one of the most important viral pathogens of livestock and causes substantial economic losses to the livestock industry worldwide. BVDV is not necessarily species specific and is known to infect domesticated and wild ruminants. In the present study, BVDV infection was identified in two Saanen goats from one farm, and two different viral subtypes were found, BVDV-1a and BVDV-2a. Each isolate was closely related to cattle isolates identified in the Republic of Korea. The two sequences obtained in this study were not consistent with border disease virus (BDV). The incidence of BVDV in this farm apparently occurred in the absence of contact with cattle and may be associated with grazing. This study demonstrates that BVDV infection may be possible to transmit among goats without exposure to cattle. Therefore, this result indicates that Saanen goats may act as natural reservoirs for BVDV. This is the first report of BVDV-1a infection in a Saanen goat.

STAT5 plays a critical role in regulating the 5'-flanking region of the porcine whey acidic protein gene in transgenic mice.

The mammary gland serves as a valuable bioreactor system for the production of recombinant proteins in lactating animals. Pharmaceutical-grade recombinant protein can be harvested from the milk of transgenic animals that carry a protein of interest under the control of promoter regions genes encoding milk proteins. Whey acidic protein (WAP), for example, is predominantly expressed in the mammary gland and is regulated by lactating hormones during pregnancy. We cloned the 5'-flanking region of the porcine WAP gene (pWAP) to confirm the sequence elements in its promoter that are required for gene-expression activity. In the present study, we investigated how lactogenic hormones--including prolactin, hydrocortisone, and insulin--contribute to the transcriptional activation of the pWAP promoter region in mammalian cells, finding that these hormones activate STAT5 signaling, which in turn induce gene expression via STAT5 binding sites in its 5'-flanking region. To confirm the expression and hormonal regulation of the 5'-flanking region of pWAP in vivo, we generated transgenic mice expressing human recombinant granulocyte colony stimulating factor (hCSF2) in the mammary gland under the control of the pWAP promoter. These mice secreted hCSF2 protein in their milk at levels ranging from 242 to 1,274.8 ng/ml. Collectively, our findings show that the pWAP promoter may be useful for confining the expression of foreign proteins to the mammary gland, where they can be secreted along with milk.

Two cases of meningocele and meningoencephalocele in Jeju native pigs.

BACKGROUND: Meningocele and meningoencephalocele of the skull are congenital deformities. Various species, such as pigs, dogs, and cats, are susceptible to congenital meningocele and meningoencephalocele and the incidence is higher in large white and landrace pigs. CASE PRESENTATION: In this study, swelling was observed in the fontanel areas of the median planes of the skull cap in two female piglets of the same litter. Gross clinical examination, neurological examination, computed tomography (CT), and magnetic resonance imaging (MRI) were conducted on the symptomatic piglets. The gross clinical and neurological examinations revealed no specific findings, except for the swellings. According to the CT results, the length of the defect on the sagittal section of the skull was 4.7 mm in case 1 and 20.62 mm in case 2. Connected flow between the skull swellings and the cerebrospinal fluid (CSF) of the lateral ventricles was observed, and partial herniation was identified in case 2. On MRI, CSF with high T2 signals was identified in the arachnoid spaces between the cerebrum and the cerebellum in the two cases, which is consistent with intracranial hypertension. The size of the swelling formed in the parietal bones was 1.6 × 1.1 × 1.8 cm(3) (case 1) and 1.2 × 1.38 × 1.7 cm(3) (case 2). The increase in intracranial pressure was more obvious in case 2 than in case 1, and was accompanied by posterior displacements of the mesencephalon and cerebellum. CONCLUSIONS: Case 1 was diagnosed as meningocele resulting from meningeal herniation and case 2 was diagnosed as meningoencephalocele caused by brain tissue herniation.

Cell-free extract from porcine induced pluripotent stem cells can affect porcine somatic cell nuclear reprogramming.

Pretreatment of somatic cells with undifferentiated cell extracts, such as embryonic stem cells and mammalian oocytes, is an attractive alternative method for reprogramming control. The properties of induced pluripotent stem cells (iPSCs) are similar to those of embryonic stem cells; however, no studies have reported somatic cell nuclear reprogramming using iPSC extracts. Therefore, this study aimed to evaluate the effects of porcine iPSC extracts treatment on porcine ear fibroblasts and early development of porcine cloned embryos produced from porcine ear skin fibroblasts pretreated with the porcine iPSC extracts. The Chariot(TM) reagent system was used to deliver the iPSC extracts into cultured porcine ear skin fibroblasts. The iPSC extracts-treated cells (iPSC-treated cells) were cultured for 3 days and used for analyzing histone modification and somatic cell nuclear transfer. Compared to the results for nontreated cells, the trimethylation status of histone H3 lysine residue 9 (H3K9) in the iPSC-treated cells significantly decreased. The expression of Jmjd2b, the H3K9 trimethylation-specific demethylase gene, significantly increased in the iPSC-treated cells; conversely, the expression of the proapoptotic genes, Bax and p53, significantly decreased. When the iPSC-treated cells were transferred into enucleated porcine oocytes, no differences were observed in blastocyst development and total cell number in blastocysts compared with the results for control cells. However, H3K9 trimethylation of pronuclear-stage-cloned embryos significantly decreased in the iPSC-treated cells. Additionally, Bax and p53 gene expression in the blastocysts was significantly lower in iPSC-treated cells than in control cells. To our knowledge, this study is the first to show that an extracts of porcine iPSCs can affect histone modification and gene expression in porcine ear skin fibroblasts and cloned embryos.

A Horsehair Worm, Gordius sp. (Nematomorpha: Gordiida), Passed in a Canine Feces.

Nematomorpha, horsehair or Gordian worms, include about 300 freshwater species in 22 genera (Gordiida) and 5 marine species in 1 marine genus (Nectonema). They are parasitic in arthropods during their juvenile stage. In the present study, the used gordian worm was found in the feces of a dog (5-month old, male) in July 2014. Following the worm analysis using light and scanning electron microscopes, the morphological classification was re-evaluated with molecular analysis. The worm was determined to be a male worm having a bi-lobed tail and had male gonads in cross sections. It was identified as Gordius sp. (Nematomorpha: Gordiidae) based on the characteristic morphologies of cross sections and areole on the cuticle. DNA analysis on 18S rRNA partial sequence arrangements was also carried out, and the gordiid worm was assumed to be close to the genus Gordius based on a phylogenic tree analysis.

Detection of Tick-Borne Pathogens in the Korean Water Deer (Hydropotes inermis argyropus) from Jeonbuk Province, Korea.

The objective of this study was to investigate the prevalence of tick-borne pathogens in the Korean water deer (Hydropotes inermis argyropus). Pathogens were identified using PCR which included Anaplasma, Ehrlichia, Rickettsia, and Theileria. Rickettsia was not detected, whereas Anaplasma, Ehrlichia, and Theileria infections were detected in 4, 2, and 8 animals, respectively. The most prevalent pathogen was Theileria. Of the 8 Theileria-positive animals, 2 were mixed-infected with 3 pathogens (Anaplasma, Ehrlichia, and Theileria) and another 2 animals showed mixed-infection with 2 pathogens (Anaplasma and Theileria). Sequencing analysis was used to verify the PCR results. The pathogens found in this study were identified as Anaplasma phagocytophilum, Ehrlichia canis, and Theileria sp. To the best of our knowledge, this is the first report identifying these 3 pathogens in the Korean water deer. Our results suggest that the Korean water deer may serve as a major reservoir for these tick-borne pathogens, leading to spread of tick-borne diseases to domestic animals, livestock, and humans. Further studies are needed to investigate their roles in this respect.

Detection of Anaplasma sp. in Korean Native Goats (Capra aegagrus hircus) on Jeju Island, Korea.

Anaplasma species are obligate intracellular pathogens that can cause tick-borne diseases in mammalian hosts. To date, very few studies of their occurrence in Korean native goats (Capra aegagrus hircus) have been reported. In the present study, we investigated Anaplasma infection of Korean native goats on Jeju Island, Republic of Korea, and performed phylogenetic analysis based on the 16S rRNA gene sequences. Our results showed that Anaplasma infection was found mostly in adult female goats. The phylogenetic tree revealed that the 7 sequences identified in Korean native goats could belong to Anaplasma sp. and were distinct from A. marginale, A. centrale, and A. ovis. The results indicated that the sequences identified to belong to Anaplasma were closely related to sequences isolated from goats in China and were clustered within the same group. To our knowledge, this is the first study to detect Anaplasma sp. infection in Korean native goats.

Mineral patterns in hair: A decisive factor between reproducible and repeat breeder dairy cows.

Reproduction, especially impregnation, is a critical aspect of dairy cow management that directly influences herd milk productivity. We conducted a noninvasive hair mineral assay to compare the mineral profiles of two dairy cow groups: reproducible and repeat breeder, by investigating the levels of 11 essential minerals (Ca, Mg, Na, K, Fe, Cu, Mn, Zn, Cr, Se, and P) and 6 toxic elements (Hg, Pb, Cd, Al, As, and Ni) in both groups. We also conducted principal component and correlation matrix analyses to compare hair mineral patterns between the groups. Compared to their reproducible counterparts, repeat breeder cows had lower levels of Na, K, and Se. However, Fe, Cd, Al, and As levels were higher in repeat breeders than in their reproducible counterparts. The correlation matrix showed notable correlation patterns for each group. Ca, K, and Na levels were positively correlated in reproducible cows, whereas repeat breeder cows showed positive correlations only between Ca and K levels. Se showed positive correlations with Zn only in the reproducible cow group. Negative correlations were not found in the reproducible group, whereas the repeat breeder group exhibited 7 negative correlations. Despite the limitations of hair mineral analysis, this study provided useful insights into the reproductive potential of dairy cows. These findings aid in easing the prediction of repeat breeder occurrences in herds and are expected to facilitate timely mineral supplementation and other interventions to improve overall herd reproduction in dairy farms.

Comparing Gut Microbial Composition and Functional Adaptations between SPF and Non-SPF pigs.

The gut microbiota is closely associated with digestion, metabolism, immunity, and host health. The imbalance of the microbial community in livestock directly affects their well-being and, consequently, productivity. The composition and diversity of the gut microbiota are influenced not only by host genetics but also by environmental factors such as the microbial complexity of the rearing environment, feeds, and antibiotics. Here, we focus on the comparison of gut microbial communities in miniature pigs developed for xenotransplantation in specific pathogen-free (SPF) and conventional (non-SPF) facilities. To identify the disparities in gut microbial composition and functionality between these two environments, 16S RNA metagenome sequencing was conducted using fecal samples. The results revealed that the non-SPF pigs had higher gut microbiota diversity than the SPF pigs. The genera Streptococcus and Ruminococcus were more abundant in SPF pigs than in non-SPF pigs. Blautia, Bacteroides, and Roseburia were exclusively observed in SPF pigs, whereas Prevotella was exclusively found in non-SPF pigs. Carbohydrate and nucleotide metabolism, as well as environmental information processing, were predicted to be enriched in SPF pigs. In addition, energy and lipid metabolism, along with processes related to genetic information, cellular communication, and diseases, were predicted to be enriched in non-SPF pigs. This study makes an important contribution to elucidating the impact of environments harboring a variety of microorganisms, including pathogens, on the gut microbiota of miniature pigs. Furthermore, we sought to provide foundational data on the characteristics of the gut microbiota in genetically modified pigs, which serve as source animals for xenotransplantation.

Application of Enzyme-Linked Fluorescence Assay (ELFA) to Obtain In Vivo Matured Dog Oocytes through the Assessment of Progesterone Level.

Successful dog cloning requires a sufficient number of in vivo matured oocytes as recipient oocytes for reconstructing embryos. The accurate prediction of the ovulation day in estrus bitches is critical for collecting mature oocytes. Traditionally, a specific serum progesterone (P4) range in the radioimmunoassay (RIA) system has been used for the prediction of ovulation. In this study, we investigated the use of an enzyme-linked fluorescence assay (ELFA) system for the measurement of P4. Serum samples of estrus bitches were analyzed using both RIA and ELFA, and the measured P4 values of ELFA were sorted into 11 groups based on the standard concentration measured in RIA and compared. In addition, to examine the tendency of changes in the P4 values in each system, the P4 values on ovulation day (from D - 6 to D + 1) in both systems were compared. The ELFA range of 5.0-12.0 ng/mL was derived from the RIA standard range of 4.0-8.0 ng/mL. The rates of acquired matured oocytes in RIA and ELFA were 55.47% and 65.19%, respectively. The ELFA system successfully produced cloned puppies after the transfer of the reconstructed cloned oocytes. Our findings suggest that the ELFA system is suitable for obtaining in vivo matured oocytes for dog cloning.

Plum-Derived Exosome-like Nanovesicles Induce Differentiation of Osteoblasts and Reduction of Osteoclast Activation.

Osteoblasts and osteoclasts play crucial roles in bone formation and bone resorption. We found that plum-derived exosome-like nanovesicles (PENVs) suppressed osteoclast activation and modulated osteoblast differentiation. PENVs increased the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells and osteoblasts from mouse bone marrow cultures. Notably, PENVs elevated the expression of osteoblastic transcription factors and osteoblast differentiation marker proteins in MC3T3-E1 cells. Higher levels of phosphorylated BMP-2, p38, JNK, and smad1 proteins were detected in PENV-treated MC3T3-E1 cells. Additionally, the number of TRAP-positive cells was significantly decreased in PENV-treated osteoclasts isolated from osteoblasts from mouse bone marrow cultures. Importantly, osteoclastogenesis of marker proteins such as PPAR-gamma, NFATc1, and c-Fos were suppressed by treatment with PENVs (50 µg/mL). Taken together, these results demonstrate that PENVs can be used as therapeutic targets for treating bone-related diseases by improving osteoblast differentiation and inhibiting osteoclast activation for the first time.

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.].

Establishment of intestinal organoids from small intestine of growing cattle (12 months old).

Recently, we reported the robust in vitro three-dimensional (3D) expansion of intestinal organoids derived from adult bovine (> 24 months) samples. The present study aimed to establish an in vitro 3D system for the cultivation of intestinal organoids derived from growing cattle (12 months old) for practical use as a potential alternative to in vivo systems for various purposes. However, very few studies on the functional characterization and 3D expansion of adult stem cells from livestock species compared to those from other species are available. In this study, intestinal crypts, including intestinal stem cells, from the small intestines (ileum and jejunum) of growing cattle were isolated and long-term 3D cultures were successfully established using a scaffold-based method. Furthermore, we generated an apical-out intestinal organoid derived from growing cattle. Interestingly, intestinal organoids derived from the ileum, but not the jejunum, could be expanded without losing the ability to recapitulate crypts, and these organoids specifically expressed several specific markers of intestinal stem cells and the intestinal epithelium. Furthermore, these organoids exhibited key functionality with regard to high permeability for compounds up to 4 kDa in size (e.g., fluorescein isothiocyanate [FITC]-dextran), indicating that apical-out intestinal organoids are better than other models. Collectively, these results indicate the establishment of growing cattle-derived intestinal organoids and subsequent generation of apical-out intestinal organoids. These organoids may be valuable tools and potential alternatives to in vivo systems for examining host-pathogen interactions involving epithelial cells, such as enteric virus infection and nutrient absorption, and may be used for various purposes.