The distribution pattern of collagen fibril diameter in the equine superficial digital flexor tendon (SDFT) is known to differ in central and peripheral areas of some regions. This study reports the essence of collagen fibril differences among different regions of the equine SDFT by transmission electron microscopic (TEM) and high-voltage electron microscopic observations and biochemical analysis. The distribution of large collagen fibrils increased but the density of collagen fibrils decreased from the proximal metacarpal region to the distal metacarpal region. Large collagen fibrils with an irregular cross-sectional profile were found more frequently in the middle metacarpal region than in other regions. Three-dimensional reconstruction of images of irregularly shaped collagen fibrils revealed that these fibrils are formed through fusion of small collagen fibrils with large ones. The amount of decorin, which reportedly inhibits the lateral fusion of collagen fibrils, decreased in the direction of the distal metacarpal region. On the other hand, the size of decorin gradually increased in the direction of the distal metacarpal region. These results suggest that regional differences in collagen fibril distribution and density of collagen fibrils in the SDFT are due, at least in part, to fusion of collagen fibrils and the concomitant regional differences in the amount and size of decorin.
Although we recently clarified sex-based differences in mouse kidney morphology, the developmental processes responsible for these gender differences during maturation remain unclear. The present study analyzed the morphometry of kidneys from 20-, 30-, 50-, 60-, 70-, 90-, 120- and 150-day-old DBA/2Cr mice. Total kidney weight and ratio of kidney weight to body weight were larger in males than females beginning at 50 days of age. The percentage of renal corpuscles exhibiting a cuboidal parietal layer was higher in males than in females in the 70-day and older mice. The diameter of cortical renal corpuscles was larger in males than in females beginning on day 90. The number of proximal convoluted tubular cell nuclei was higher in females than in males from day 90 onward. Vacuolar structures in the proximal convoluted tubular epithelium became prominent in 70-day-old males. PAS-positive granules in the proximal straight tubular epithelium became prominent in females on day 50. This paper is the first to describe the development of gender differences in mouse kidney morphology.
Anemia is a major secondary symptom in chronic renal disorder (CRD), but the precise cause of insufficient production of erythropoietin (EPO) remains unclear owing to the controversial localization of EPO-producing cells in the kidneys. The ICR-derived glomerulonephritis (ICGN) mouse, a new hereditary nephrotic mouse, is an appropriate model of anemia associated with CRD. By using an amplified in situ hybridization technique, we detected and counted the renal EPO-producing cells under both normoxic and hypoxic conditions. The expression levels of renal EPO mRNA were quantified and oxygen gradients were also assessed immunohistochemically. Amplified in situ hybridization clarified that EPO-producing cells were peritubular interstitial cells in the middle region of renal cortex in both ICR and ICGN mice. Hypoxia (7% O2) induced low oxygen tension in proximal tubular epithelial cells of renal cortex, and increased the expression of EPO mRNA and the number of EPO-producing cells in both ICR and ICGN mice. However, hypoxia did not increase the serum EPO levels in ICGN mice. The ICGN mouse is a good model for anemia associated with CRD, and the suppression of EPO protein production in the renal EPO-producing cells is considered to be a potential cause of anemia associated with CRD.
To investigate the distribution of the early stage chondrocytes during the formation and closure of epiphyseal growth plate (EGP) of the domestic cat, we examined the EGP of proximal tibiae by immunohistochemistry for type VI collagen. In the epiphyseal cartilage without the secondary ossification center (SOC) and EGP in newborn cats aged 1 and 10 days, type VI collagen-positive chondrocytes were located around the cartilage canals and articular surface. In the epiphyseal cartilage with the SOC and EGP in young cats aged 1 to 3 months, type VI collagen-positive chondrocytes were located in the upper resting zone of the EGP, and then increased throughout the resting zone along with maturation. In the adult cats with the partially closed EGP, type VI collagen-positive chondrocytes were distributed throughout the remaining EGP. These findings indicate that the early stage chondrocytes characterized with type VI collagen are continuously located in the EGP during maturation. In addition, the increase of the early stage chondrocytes and the decrease of the reserve chondrocytes in the EGP along with maturation may cause the cessation of the longitudinal growth of the EGP, and finally bring about the EGP closure.
Eimeria necatrix-specific ELISA, using a recombinant antigen (the cDNA-clone NP19 expressing protein), was utilized to detect antibodies against E. necatrix in breeder pullet flocks that had previously received an attenuated live vaccine to E. necatrix. Vaccinated flocks were discriminated significantly from non-vaccinated flocks by their antibody titers and antibody positive rates at 30-55 days post-vaccination. In addition, E. necatrix-oocysts were confirmed in fecal samples of vaccinated flocks using PCR in the case where the antibody positive rates rose. These findings implied that the vaccination prompted repeated infections, and consequently the chickens generated antibodies and secured their protection against virulent field-E. necatrix. Therefore, the ELISA was suggested to be a useful tool to estimate the immune state of chickens as a result of vaccination with a live E. necatrix-vaccine.
Visual evoked potentials by flash stimulation (flash VEP) were analyzed in dogs using a topographic method. The flash VEP consisted of 3 positive (P1, P2 and P3) and 2 negative (N1 and N2) components by 150 msec after the flash stimuli. On the topographic mappings, a negative response area was observed in the frontal region of the scalp in the stimulated site followed by the shifting of the area to the contralateral frontal region and occipital region, during the first 100 msec. The negative response area in the frontal region in the stimulated site, contralateral frontal and temporal region, and occipital region were corresponded to N1, P2, and N2 on the flash VEP, respectively, according to their latencies. In the dogs with experimentally impaired the right lateral geniculate body, the latency of P2 was prolonged, and N2 and P3 were disappeared after the left eye stimulation. On the topographic mapping, only the early negative response area was detected on the stimulated site of the frontal region of the brain. Therefore, it is concluded that P1 and N1, P2, and N2 are referred to the retinal potentials, the potentials from the retina to the brainstem included the lateral geniculate body, and those from the brainstem to the visual cortex, respectively.
Dopamine and noradrenaline are catecholamine neurotransmitters that are produced by biosynthetic enzymes such as tyrosine hydroxylase (TH) and dopamine β -hydroxylase (DBH). As a first step to elucidate the genetic background of canine behavioral traits, we selected these genes as targets and sequenced these canine genes, and found that both were highly homologous with those of human beings. Then brain cDNAs derived from ten unrelated Beagles were used to search for polymorphisms in these genes. Four single nucleotide polymorphisms (SNPs) (C97T, G168A, G180A and C264T), one of which (C97T) will cause amino acid substitution in the TH gene, and two SNPs (C789A and A1819G), both of which will cause amino acid substitutions in the DBH gene were identified. The allelic frequencies among five dog breeds (47 Golden Retrievers, 41 Labrador Retrievers, 40 Malteses, 26 Miniature Schnauzers, and 39 Shibas) were examined and found to have significant variation between them with regards to all these SNPs, except for C97T in the TH gene and A1819G in the DBH gene. The polymorphisms of C97T and A1819G were found only in the Shiba. The present results suggest that the polymorphisms of the genes encoding catecholamine biosynthetic enzymes may become important markers for examining the genetic background of behavioral characteristics in dogs.
We examined in vivo efficiency of a gelatin film sheet with epidermal growth factor (EGF) for a novel therapeutic device for cutaneous wound repair. NIH3T3 fibroblasts and PAM212 keratinocytes proliferated when the cells were incubated with the homogenate of EGF containing gelatin sheets, indicating that the gelatin sheet retained biologic activity of EGF in the process of sheet formation. To evaluate therapeutic effects, the EGF containing gelatin sheets or control sheets were applied onto partial-thickness skin wounds made on dorsa of hairless dogs. Wound closure in wounds treated with EGF containing gelatin sheets was accelerated when compared to that of wounds treated with control sheets, exhibiting earlier reepithelialization of the epidermis and highly regulated repair of extracellular matrix in the dermis. Therefore, we concluded that the gelatin film is a useful material to keep EGF stable and the EGF containing sheet has the ability to become an efficient therapeutic agent for superficial or deep partial-thickness wounds in the skin.
Although the possible involvement of neurotrophic factors in itchy skins of atopic dermatitis has been predicted, the exact mechanism by which itch is induced remains unclear. Since nerve growth factor (NGF) has crucial effects on development and functions of sensory nerves, we determined production of NGF and extension of nerve fibers in skins of NC/NgaTnd mice with or without atopic dermatitis. NC/NgaTnd mice spontaneously develop atopic dermatitis-like skin lesions when they are raised in air-unregulated conventional circumstances. We quantified scratching behavior of NC/NgaTnd mice during the development of dermatitis using a novel analytical system and compared to clinical skin severity scores. A significant correlation between the severity of dermatitis and the increase in the number of scratches was identified, indicating that scratching behavior may associate with clinical skin conditions. NGF contents in the skin lesions of conventional NC/NgaTnd mice were significantly higher than those in SPF mice. Positive reactions for NGF were observed in keratinocytes and fibroblasts in affected skins of conventional NC/NgaTnd mice. Immunohistochemical analysis showed the extension of protein gene product 9.5-positive nerve fibers from the dermis toward the epidermis at the skin lesions. These results suggest that sensory nerves induced by NGF may contribute to development of itch, and that NGF produced at the affected site may provide abnormal skin sensitivity in atopic dermatitis.
We cloned, sequenced and analyzed the cDNAs encoding Camelidae inflammatory cytokines, including llama (lama glama) interleukin (IL)-1α, IL-1β, IL-6, tumor necrosis factor (TNF)-α and camel (Camelus bactrianus) IL-6 and TNF-α. The similarity levels of the deduced amino acid sequences of IL-1α, IL-1β, IL-6 and TNF-α from llama (camel) to those from other mammalian species, ranged from 60.7% to 87.7%, 52.8% to 75.3%, 41.4% to 98.6%, and 72.9% to 99.6%, respectively. Phylogenetic analyses based on nucleic acid sequences showed that llama IL-1α, IL-1β, IL-6 and TNF-α were more closely related to those of camel, pig, cattle, sheep and horse than to those of human, dog, cat, mouse and rat.
Many animals including cattle can synthesize vitamin C from glucose. The objective of this study was to investigate plasma vitamin C concentration in ketotic cows during the early lactation period because glucose supply for vitamin C synthesis might be limited in these cows. We measured plasma β -hydroxybutyrate (BHBA) concentration in 118 cows within 2 months after parturition. Subclinical/clinical ketosis was quantitatively determined using a plasma BHBA threshold of 1,200 μM. Plasma glucose concentration was lower in the ketotic cows than in the control cows but plasma vitamin C concentration did not differ between the control and the ketotic cows. Then we measured plasma vitamin C, BHBA and glucose levels in 7 cows during the periparturient period. Plasma BHBA increased and plasma glucose decreased after parturition but plasma vitamin C did not change. These results indicate that plasma vitamin C is not related to the incidence of ketosis in the early lactation period. We suggest that ketotic cows have the ability to produce vitamin C to meet its requirement in the early lactation period although glucose supply is not sufficient for milk production. Vitamin C synthesis is possibly given a high metabolic-priority for glucose in lactating cows.
A seven-year-old, spayed female mongrel dog was diagnosed as pemphigus foliaceus (PF) by clinical, histopathological and immunopathological observations. Serum antibodies against the cell surface of keratinocytes in the dog were detected by indirect immunofluorescence (IIF) using cryosectioned bovine esophagus as well as living cultured-canine keratinocytes as the substrates. When we compared the titers of IIF on bovine esophagus with its disease activity, the IIF titers reflected the disease activity throughout the time course. Our findings will suggest that sequential titration of serum antibodies by IIF will be useful for monitoring the serological disease activity in canine PF.
Thymectomy on day 3 after birth (D3Tx) is understood to eliminate CD4+CD25+ regulatory T cells (Treg) from the peripheral T cell repertoire in rodents, leading to the activation of autoreactive T cells. Herein, D3Tx was performed in IQI/Jic mice, a model for Sjögren's syndrome characterized by autoimmune infiltrations into the lacrimal and salivary glands. At the age of 16 weeks, very severe lesions were observed in lacrimal tissues from thymectomized mice, suggesting that Treg preserve their immunoregulatory function in young IQI/Jic mice. In contrast, salivary lesions were comparable in the D3Tx and control groups, raising the possibility that either salivary-specific Treg escaped elimination in thymectomized mice or spontaneous lesions in IQI/Jic mice developed independently of the tolerance through Treg.
Yersiniosis was prevalent among a caprine herd during the late autumn of 2003 in Iwate Prefecture, Japan. The disease affected 29 of about 100 lactating goats, but not dried or nonparous goats, mature male goats or kids. Four animals died within an epidemic period of 20 days. Affected animals developed decreased milk production with subsequent watery diarrhea, neutrophilia with increased band forms and multiple microabscesses characteristic of yersiniosis in the intestinal mucosa from the jejunum to caecum as well as in the mesenteric lymph nodes. Y. pseudotuberculosis serotype III was isolated from intestinal contents and mesenteric lymph nodes. The organism was also cultured from clinically normal dried animals. The outbreak might have been precipitated by multiple stress factors, such as lactation, cold weather, Corynebacterium pseudotuberculosis infection resulting in abscess formation and tapeworm and coccidium parasitisms.
A 6-year-old female ferret had a firm mass 2 cm in diameter in the pyloric region of the stomach. Histopathologically, the mass was composed of neoplastic proliferation of well-differentiated epithelial cells, showing tubular or glandular growth patterns. Osseous metaplastic foci were often found in the tumor. Tumor cells showed a positive reaction for immunohistochemistry against bone morphogenetic protein-6, an osteogenic factor. A diagnosis of gastric adenocarcinoma with ossification was made.
Babesia rodhaini is a rodent hemoparasite closely related to B. microti, the major causative agent of human babesiosis. We tested the infectivity of B. rodhaini for human erythrocytes by using the SCID mouse model in which the circulating erythrocytes were replaced with those of humans. Initially, parasites grew very poorly in the mouse model, but a variant capable of propagating in human erythrocytes emerged after an adaptation period of three weeks. In an attempt to identify parasite proteins involved in the alteration of host cell preference, an expression cDNA library of B. rodhaini was constructed and screened with immune mouse sera. Although we were able to obtain three merozoite surface protein (MSP) genes, sequences of these genes from both the parental strain and human erythrocyte-adapted substrain were identical. Our results suggest that B. rodhaini has potential ability to infect human erythrocytes, but development of this ability may not be brought about by an amino acid change in MSPs.
Some public areas in Japan such as parks and gardens can be highly contaminated with pigeon feces. We examined levels of four bacterial contaminations in fecal samples from feral pigeons in 7 prefectures. We isolated Salmonella Typhimurium and S. Cerro from 17 (3.9%) of 436 samples, as well as Mycobacterium spp. including M. avium-intracellulare complex from 29 (19.0%) of 153 samples. The polymerase chain reaction detected Chlamydia psittaci and C. pecorum in 106 (22.9%) of 463 samples, but E. coli O-157 was not isolated from any of the samples. Our results indicate that pigeon feces are a source of several zoonotic agents for birds, animals and humans.
To investigate the adverse effects of long-term administration of ketoprofen in dogs, ketoprofen (1 mg/kg) was administered to five clinically healthy beagle dogs (ketoprofen group) and gelatin capsules (control group) were administered to four clinically healthy beagle dogs for 30 days. We monitored the dogs through periodic physical examination, blood analyses, endoscopic examinations, fecal occult blood tests, renal function tests, urinalysis, urinary enzyme indices and cuticle bleeding time analysis. The lesions in the stomach, especially in the pyloric antrum, and fecal occult blood progressively worsened in the ketoprofen group. However, the differences between the ketoprofen group and the control group were not statistically significant. One dog in the ketoprofen group temporarily exhibited a decrease in renal plasma flow and two dogs exhibited enzymuria. However, these changes did not persist and the other examinations showed no significant difference between premedication and postmedication in the ketoprofen group. Therefore, the adverse effects of long-term administration of ketoprofen observed in this study were not clinically important in healthy dogs. Nevertheless, further investigation of adverse renal effects from long-term administration of ketoprofen is necessary in the dogs with subclinical renal disease.
A seven-year-old, female, domestic short hair cat was presented with a history of chronic anorexia. Radiographic examination revealed a large space-occupying calcified mass in the abdominal cavity. The mass was located in pylorus and did not extend into the duodenum and surrounding tissues. Billroth I gastroduodenostomy was conducted to remove the mass. Histopathological examination of the mass showed a lymphoma. Although Recovery following the operation was excellent, the patient showed intermittent vomiting unrelated to feeding. Radiographical examination revealed a megaesophagus, which was assumed to be a complication of the Billroth I procedure, since the condition was not observed before the procedure.