Expression and basic biochemical characteristics of recombinant surfactant protein D of bottlenose dolphin (Tursiopstruncatus).

We previously identified surfactant protein D (SP-D) in the bottlenose dolphin Tursiops truncatus as a unique evolutionary factor of the cetacean pulmonary immune system. In this short report, recombinant SP-D of bottlenose dolphin (dSP-D) was synthesized in mammalian cells, and its properties were analyzed in vitro. The recombinant proteins were purified using Ni-carrier or Co-carrier. Sodium dodecyl sulfate poly-acrylamide gel electrophoresis and western blotting revealed a 50 kDa major band with minor secondary bands. Enzyme-linked immunosorbent assay-like methods revealed that recombinant dSP-D bonded to gram-positive and gram-negative bacterial walls. Our findings suggest the clinical usefulness of dSP-D for cetacean pneumonia.

Genetic characterization of bovine respiratory syncytial viruses in Japan between 2017 and 2019.

Bovine respiratory syncytial virus (BRSV) strains that were detected in Kagoshima prefecture and isolated in Hokkaido between 2017 and 2019, together with a BRSV vaccine strain, were subjected to full-genome sequencing. The BRSV strains identified in Japan were found to be genetically close to each other but distant from the vaccine strains. The deduced amino acids at positions 206 and 208 of the glycoprotein (G protein), which form one of the major epitopes of the recent Japanese BRSV strains, were different from those of the vaccine strains. Therefore, the recent Japanese BRSV strains might be antigenically different from the BRSV vaccine strains.

Osteochondral regeneration of the femoral medial condyle by using a scaffold-free 3D construct of synovial membrane-derived mesenchymal stem cells in horses.

BACKGROUND: Medical interventions for subchondral bone cysts in horses have been extensively studied. This study investigated the regeneration of articular cartilage and subchondral bone with scaffold-free three-dimensional (3D) constructs of equine synovial membrane-derived mesenchymal stem cells (SM-MSCs) isolated from three ponies and expanded until over 1.0 × 107 cells at passage 2 (P2). RESULTS: SM-MSCs were strongly positive for CD11a/CD18, CD44, and major histocompatibility complex (MHC) class I; moderately positive for CD90, CD105, and MHC class II; and negative for CD34 and CD45 on flow cytometry and differentiated into osteogenic, chondrogenic, and adipogenic lineages in the tri-lineage differentiation assay. After culturing SM-MSCs until P3, we prepared a construct (diameter, 6.3 mm; height, 5.0 mm) comprising approximately 1920 spheroids containing 3.0 × 104 cells each. This construct was confirmed to be positive for type I collagen and negative for type II collagen, Alcian blue, and Safranin-O upon histological analysis and was subsequently implanted into an osteochondral defect (diameter, 6.8 mm; depth, 5.0 mm) at the right femoral medial condyle. The contralateral (left femoral) defect served as the control. At 3 and 6 months after surgery, the radiolucent volume (RV, mm3) of the defects was calculated based on multiplanar reconstruction of computed tomography (CT) images. Magnetic resonance (MR) images were evaluated using a modified two-dimensional MR observation of cartilage repair tissue (MOCART) grading system, while macroscopic (gross) and microscopic histological characteristics were scored according to the International Cartilage Repair Society (ICRS) scale. Compared to the control sites, the implanted defects showed lower RV percentages, better total MOCART scores, higher average gross scores, and higher average histological scores. CONCLUSIONS: Implantation of a scaffold-free 3D-construct of SM-MSCs into an osteochondral defect could regenerate the original structure of the cartilage and subchondral bone over 6 months post-surgery in horses, indicating the potential of this technique in treating equine subchondral bone cysts.

Genetic analyses for conservation of the traditional Tokara horse using 31 microsatellite markers.

In order to promote conservation of the traditional Tokara horse in its remaining three breeding areas in Japan (Nakanoshima, Kaimondake, and Iriki), we genotyped 123 horses using 31 microsatellite markers and determined their genetic diversity. On average, the number of alleles (NA), observed heterozygosity (HO), expected heterozygosity (HE), and inbreeding coefficient (FIS) among all horses were 3.0, 0.424, 0.481, and 0.108, respectively. Compared with other endangered horse breeds, we found that, even though the size of the Tokara horse population has recently increased, the NA, HO, and HE of Tokara horses are still notably lower than those of other breeds. Neighbor-joining tree and STRUCTURE analysis showed that the current population of Tokara horses is divided into three subpopulations, corresponding to their respective feeding and breeding areas: Nakanoshima, Kaimondake, and Iriki. This subdivision was also reflected in the NA of microsatellite DNAs, with four, three, and four different loci showing single alleles in Nakanoshima, Kaimondake, and Iriki horses, respectively. These alleles are considered to have become fixed as a consequence of breeding within the limited number of horses in each area. Since Tokara horses are currently strongly divided into subpopulations, it is vitally important to exchange several horses among their different breeding units in order to maintain the genetic diversity of the Tokara horse as a unique breed. The data obtained in this study contribute toward explaining the history of Tokara horses and also provide important information for future monitoring of population diversity and guiding conservation measures for this endangered breed.

A pilot study of regenerative therapy by implanting synovium-derived mesenchymal stromal cells in equine osteochondral defect models.

Synovium-derived mesenchymal stromal cells (SM-MSCs) from seven Thoroughbreds with naturally occurring intra-articular fracture proliferated to over ten million cells by the second passage. Using three experimental Thoroughbreds, columnar osteochondral defects were made arthroscopically at the bilateral distal radius. Five million allogenic SM-MSCs were implanted into the right defect, and another five million were injected into the right radio-carpal joint (implantation site). No SM-MSCs were implanted into the left defect or the same joint (control site). At 3 and 6 weeks after surgery, ten million autologous SM-MSCs were injected into the right joints. Radiolucent volumes of defects calculated by analysis of postmortem CT images 9 weeks after surgery were decreased in implanted sites compared with control sites in all horses. The average scores for ICRS gross and histopathological grading scales in implanted sites were equal to or higher than those of the controls. These results suggest that allogenic implantation and subsequent autologous injection of SM-MSCs might not obstruct subchondral bone formation in defects.

Distribution of Y chromosomal haplotypes in Japanese native horse populations.

The distribution of Y chromosomal haplotypes in Japanese native horse populations was investigated to obtain genetic information on these populations. Here, 159 male/gelded horses from eight local populations were investigated, and three Y haplotypes (JHT-1, JHT-2, and JHT-3) were identified by analyzing five Y-linked loci. Five populations had only JHT-1, whereas two populations had only JHT-2. One population had JHT-1 and JHT-3. Based on the geographical distribution of these haplotypes and previously reported haplotypes for other Asian horses, JHT-1 is considered to be a major haplotype in ancestral native horses. The fixation of each haplotype suggests the influence of independent breeding and genetic drift in each population. These findings complement the results from previous genetic studies of Japanese native horses.

Isolation of equine peripheral blood stem cells from a Japanese native horse.

The sizes of Japanese native horses have drastically decreased, and protection of these populations is important for Japanese horse culture. Social trials as well as scientific attempts are necessary for maintaining the breed. Mesenchymal stem cells (MSCs) have potential as a cell source for various cell therapies. However, there have been no reports on MSCs of Japanese native horses. We aimed to isolate and characterize MSCs from a Japanese native horse, the Noma horse. Plastic-adherent and self-replicating cells were isolated from a Noma horse's peripheral blood (PB). The isolated cells had trilineage potential and a surface antigen of mesenchymal cells, so they fulfilled the minimal criteria of MSCs. Therefore, PB can be one source of MSCs for Japanese native horses.

Pituitary pars intermedia dysfunction (equine Cushing's disease) in a Thoroughbred stallion: a single report.

Equine pituitary pars intermedia dysfunction (PPID) generally occurs in older horses showing hirsutism, delayed molting, weight loss, polydipsia, polyuria, laminitis, and reproductive disorders (in broodmares), but there have been no reports on stallions. This report presents a case of a 21-year-old Thoroughbred stallion that developed hirsutism and experienced delayed molting. There were no abnormal findings for semen quality or the stallion's sexual desire. The horse was diagnosed with PPID based on dexamethasone suppression test and plasma levels of adrenocorticotropic hormone. It was then medicated with pergolide mesylate. Since the horse died due to humerus fracture, an autopsy was conducted, and pituitary adenoma was confirmed. No pathological findings were defined in the testicles; therefore, reproductive activity might not have been impaired.

Methicillin-resistant Staphylococcus aureus ulcerative keratitis in a Thoroughbred racehorse.

We report the first case of methicillin-resistant Staphylococcus aureus (MRSA) keratitis in a racehorse. A 5-year-old mare developed punctate keratitis after racing. The corneal ulcer continued to expand despite ophthalmic antimicrobial therapy. On day 6, a conjunctival graft surgery was performed. The mare was euthanized, following colitis and laminitis development on day 10. MRSA was isolated from the corneal swab taken at the time of euthanasia. Immunohistochemical analysis demonstrated gram-positive and anti-S. aureus monoclonal antibody-positive cocci infiltration of the corneal stroma; and a diagnosis of MRSA ulcerative keratitis was made. An ophthalmic antimicrobial against the isolated MRSA did not improve the ocular lesion. The MRSA strain was found to be staphylococcal cassette chromosome mec type II, a strain frequently isolated from humans in Japan.

A study of the distribution of color Doppler flows in the superficial digital flexor tendon of young Thoroughbreds during their training periods.

Aim of this study was to evaluate the relationships of exercise and tendon injury with Doppler flows appearing in the superficial digital flexor tendon (SDFT) of young Thoroughbreds during training periods. The forelimb SDFTs of 24 one- to two-year-old Thoroughbreds clinically free of any orthopaedic disorders were evaluated using grey-scale (GS) and color Doppler (CD) images during two training periods between December 2013 to April 2015. Twelve horses per year were examined in December, February, and April in training periods that began in September and ended in April. The SDFT was evaluated in 3 longitudinal images of equal lengths (labelled 1, 2, 3 in order from proximal to distal), and 6 transversal images separated by equal lengths (labelled 1A, 1B, 2A, 2B, 3A and 3B in order from proximal to distal) of the metacarpus using both GS and CD. The running (canter and gallop) distance for 1 month before the date of the ultrasonographic examinations was increased in December, February, and April in both of the two training periods. CD flows defined as rhythmically blinking or pulsatory colored signals were found in 56 of 864 (6.4%) transversal CD images, in 28, 12, 13, and 3 images of 1A, 1B, 2A and 2B, respectively, and in 7, 14, and 35 images captured in December, February, and April, respectively. There were no longitudinal or transversal GS images indicating injury in the SDFTs in either of the two training periods. The increase of CD flows in the proximal regions of the SDFT are possibly related to the increase of the running distance during the training periods of the one- to two-year-old Thoroughbreds. Because no injury was diagnosed in the SDFTs by GS images during the training periods, the increase of CD flows in the proximal parts of SDFT is not necessarily predictive of tendon injury in the near future during the training period of young Thoroughbreds.

Comparison of the Occurrence of Transportation-associated Fever in 2 Years Old Thoroughbreds before and after Introduction of Prophylactic Marbofloxacin Administration.

In order to reveal the preventive effect of marbofloxacin (MRFX) administration just before transportation, we compared the occurrence of transportation-associated fever before and after introduction of MRFX administration. After the introduction of prophylactic MRFX administration, the rectal temperatures of horses after transportation were significantly lower than before the introduction of MRFX administration (P<0.01) and the number of febrile horses was significantly lower than before the introduction of MRFX administration (P<0.01). In conclusion, these results show that prophylactic MRFX administration just before transportation is clinically effective at preventing transportation-associated fever.

Equine keratomycosis in Japan.

OBJECTIVE: To describe the incidence, clinical progress, visual outcome, and laboratory findings of equine keratomycosis in Japan. PROCEDURE: Retrospective study of the medical records of horses clinically and mycologically diagnosed with keratomycosis at the Equine Hospitals of the Japan Racing Association from 2005 to 2011. RESULTS: The diagnosis of keratomycosis was confirmed in eight horses (40.0% of the 20 horses with infectious keratitis from which fungi and/or bacteria were isolated). Fungi recovered from corneal swabs were identified as Aspergillus flavus (4), Aspergillus niger (1), Fusarium solani (1), and Mortierella wolfii (2). All horses were treated medically with topical antifungals, and one horse was also treated surgically. The median of treatment period was 40 days. Two horses were rendered blind in the affected eye and the others retained vision. CONCLUSIONS: Equine keratomycosis comprises a considerable portion of infectious keratitis in Japan, and the causative fungi that we isolated had been isolated previously from horses with keratomycosis in other regions with the exception of M. wolfii. Culture and cytological examination of corneal lesions should be immediately performed on eyes with signs of keratitis, particularly on those not improving with antibacterial medication, as early initiation of aggressive antifungal treatment tended to result in better outcome and shorter treatment period.

Distribution of enrofloxacin within the bronchoalveolar region of healthy pigs.

This study aimed to elucidate the distribution of enrofloxacin (ERFX) within the bronchoalveolar region of pigs. Six clinically healthy pigs were allocated to intramuscular treatments with either a single dose of 5 mg/kg or 7.5 mg/kg ERFX. Samples of plasma and bronchoalveolar lavage fluid (BALF) were obtained from each pig 0 (before administration), 3, 8, and 24 hr after ERFX administration. The ERFX concentrations in pulmonary epithelial lining fluid (ELF) and BALF cells showed a similar pattern during the experimental period, whereby ERFX concentrations in both ELF and BALF cells were higher than those in the plasma. These results suggest that intramuscularly injected ERFX is well-distributed in the bronchoalveolar region.

An evolutionary medicine perspective on the cetacean pulmonary immune system - The first identification of SP-D and LBP in the bottlenose dolphin (Tursiops truncatus).

Evolutionary medicine expresses the present status of biomolecules affected by past evolutionary events. To clarify the whole picture of cetacean pneumonia, which is a major threat to cetaceans, their pulmonary immune system should be studied from the perspective of evolutionary medicine. In this in silico study, we focused on cetacean surfactant protein D (SP-D) and lipopolysaccharide-binding protein (LBP) as two representative molecules of the cetacean pulmonary immune system. Sequencing and analyzing SP-D and LBP in the bottlenose dolphin (Tursiops truncatus) lung and liver tissue collected post-mortem elucidated not only basic physicochemical properties but also their evolutionary background. This is the first study to report the sequences and expression of SP-D and LBP in the bottlenose dolphin. Besides, our findings also suggest the direction of an evolutionary arms race in the cetacean pulmonary immune system. These results have important positive implications for cetacean clinical medicine.

Pharmacokinetics in plasma and alveolar regions of a healthy calf intramuscularly administered a single dose of orbifloxacin.

This study analyzed the pharmacokinetics of orbifloxacin (OBFX) in plasma, and its migration and retention in epithelial lining fluid (ELF) and alveolar cells within the bronchoalveolar lavage fluid (BALF). Four healthy calves received a single dose of OBFX (5.0 mg/kg) intramuscularly. Post-administration OBFX dynamics were in accordance with a non-compartment model, including the absorption phase. The maximum concentration (Cmax) of plasma OBFX was 2.2 ± 0.1 µg/ml at 2.3 ± 0.5 hr post administration and gradually decreased to 0.3 ± 0.2 µg/ml at 24 hr following administration. The Cmax of ELF OBFX was 9.3 ± 0.4 µg/ml at 3.0 ± 2.0 hr post administration and gradually decreased to 1.2 ± 0.1 µg/ml at 24 hr following administration. The Cmax of alveolar cells OBFX was 9.3 ± 2.9 µg/ml at 4.0 hr post administration and gradually decreased to 1.1 ± 0.2 µg/ml at 24 hr following administration. The half-life of OBFX in plasma, ELF, and alveolar cells were 6.9 ± 2.2, 7.0 ± 0.6, and 7.8 ± 1.6 hr, respectively. The Cmax and the area under the concentration-time curve for 0-24 hr with OBFX were significantly higher in ELF and alveolar cells than in plasma (P<0.05). These results suggest that OBFX is distributed and retained at high concentrations in ELF and alveolar cells at 24 hr following administration. Hence, a single intramuscular dose of OBFX (5.0 mg/kg) may be an effective therapeutic agent against pneumonia.

Flow cytometric analysis of bronchoalveolar lavage fluid immune dynamics in calves.

Understanding the immune dynamics in the respiratory mucosa of calves is necessary for a good management of bovine respiratory disease. Immune dynamics in the respiratory mucosa in humans and experimental animals has been assessed by flow cytometric analysis of bronchoalveolar lavage fluid (BALF); however, few reports have addressed this subject in calves. The aim of this study was to establish a universal method to analyze bronchoalveolar lavage fluid (BALF) by flow cytometry and to obtain basic knowledge of bovine respiratory mucosal immune dynamics. We investigated the immune cell populations in BALF and evaluated the surface antigen expression of alveolar macrophages in calves using flow cytometer. To further analyze the surface antigen variation observed in alveolar macrophages in detail, stimulation assays were performed in vitro. BALF cells were separated into three distinct populations based on their light scatter plot, which were considered to be macrophages, lymphocytes, and neutrophils. In most individuals, most of the BALF immune cells were alveolar macrophages, but an increased proportion of lymphocytes and neutrophils was observed in some individuals. Analysis of each surface antigen expression in alveolar macrophages showed that CD21 and MHC class II expression changed in response to changes in the leukocyte population. Moreover, when alveolar macrophages were stimulated with interferon-γ in vitro, the expression of CD21 was drastically reduced and MHC class II was increased, suggesting that functional changes in alveolar macrophages themselves are involved in the immune dynamics.

Interferon gamma, lipopolysaccharide, and modified-live viral vaccines stimulation alter the mRNA expression of tumor necrosis factor α, inducible nitric oxide synthase, and interferon ß in bovine alveolar macrophages.

To understand the pathogenesis of bovine respiratory disease (BRD), it is necessary to elucidate the mechanisms of alveolar macrophage regulation by cytokines and pathogen-associated molecular patterns (PAMPs). Moreover, "non-specific effects (NSEs)" an innate immune regulatory mechanism in response to vaccines containing PAMPs, has recently attracted attention. It may be applied to BRD control, but there is limited knowledge in bovine. To investigate this, we stimulated alveolar macrophages in vitro with lipopolysaccharide (LPS), polyinosinic-polycytidylic acid sodium salt (Poly I:C), interferon gamma (IFN-γ), and modified-live viral (MLV) vaccines, respectively, and analyzed changes in tumor necrosis factor alpha (TNF-α), inducible nitric oxide synthase (iNOS), and interferon beta (IFN-ß) mRNA expression levels. mRNA expression levels of TNF-α, iNOS, and IFN-ß were significantly increased in bovine alveolar macrophages stimulated by IFN-γ and MLV vaccine; LPS, IFN-γ, and MLV vaccine; and MLV vaccine only, respectively. Additionally, all MLV vaccine-stimulated mRNA expression increases were observed in a concentration-dependent manner. These results revealed in part, the mechanism of bovine alveolar macrophage regulation by cytokines and PAMPs. Understanding the regulatory mechanisms of alveolar macrophages will contribute to understanding the pathogenesis of BRD and preventive and therapeutic BRD management based on NSEs.

Rhabdomyolysis, myoglobinuric nephrosis, and crystalline nephropathy in a captive bottlenose dolphin.

A 5-y-old female bottlenose dolphin (Tursiops truncatus) from an aquarium in Japan had clinical signs of anorexia, vomiting, and bradykinesia. Enrofloxacin and lactated Ringer solution were administered for treatment of bacterial infection and for rehydration. Elevations of creatine kinase and aspartate aminotransferase activities were detected on day 4 of treatment, indicating that rhabdomyolysis had developed on day 3. On day 5, serum creatinine and urea concentrations increased and remained high throughout the remaining treatment; the dolphin died on day 16. Postmortem examination revealed massive necrosis of the longissimus dorsi muscles. Histologic examination revealed extensive necrosis of skeletal myofibers, multifocal renal tubular necrosis with intratubular casts and crystals, and suppurative bronchopneumonia. The renal casts labeled positively with anti-myoglobin antibody; expression of aquaporin-1 was decreased in renal tubules compared to normal kidney tissue. To our knowledge, this description of clinicopathologic findings of rhabdomyolysis leading to acute kidney injury with concomitant crystalline nephropathy has not been reported previously in a bottlenose dolphin.

Mortierella wolfii keratomycosis in a horse.

PURPOSE: To describe a case of superficial keratomycosis caused by Mortierella wolfii (M. wolfii) in a horse. METHODS: A thoroughbred filly was presented with painful right eye of 2 days' duration. A superficial corneal ulcer was observed ventrally together with multifocal punctuate opacities axially. Samples were collected by swabbing and scraping the ulcerated lesion and submitted for microbiologic and cytologic examination. RESULTS: Microscopic evaluation of debrided corneal tissue revealed the presence of nonseptate fungal hyphae, and culture of a corneal swab yielded fungal growth. Medical treatment with topical antifungal, antibiotic and autogenous serum and systemic anti-inflammatory resolved the problem within 2 weeks. CONCLUSIONS: Cytologic evaluation of a corneal scraping was useful to make a clinical diagnosis of keratomycosis. Based on the mycological characteristics, the fungus isolated from the corneal lesion was identified as M. wolfii. To the authors' knowledge, this is the first case report of equine keratomycosis associated with this fungus, although the organism is known to infect various organs of cattle.

Presence of surfactant proteins in the uteri and placentae of pregnant mares.

Immunohistochemical investigations of the expression of surfactant protein A (SP-A) and surfactant protein D (SP-D) in the uterine and placental tissues of 13 pregnant mares were performed using anti-horse monoclonal primary antibodies. Strong positive reactions for both SP-A and SP-D were observed in the trophoblasts in the microcotyledons of the placentae at 182 to 314 days of gestation; in uterine glandular epithelial cells, faint-to-weak reactions were observed during gestation. This study describes, for the first time, the changes in the SP-A and SP-D expression levels in the endometrium of mares during gestation; the SP-A and SP-D expression levels increased after the second trimester of gestation.