The secretory pattern and source of immunoreactive prolactin in pregnant African (Loxodonta africana) and Asian (Elephas maximus) elephants

The objective of the present study was to define the secretion of prolactin (PRL) in pregnant African and Asian elephants. Levels of immunoreactive (ir-) PRL in serum and placental homogenates were measured by a heterologous radioimmunoassay (RIA) based on an ovine and human RIA system, and the localization of ir-PRL in the placenta was detected by immunohistochemistry using anti-human PRL. Circulating ir-PRL clearly showed a biphasic pattern during pregnancy in African and Asian elephants. Serum levels of ir-PRL started to increase from the 4 - 6th month of gestation and reached the first peak level around the 11-14th month. A second peak of circulating ir-PRL levels was observed around the 18-20th month of gestation followed by an abrupt decline after parturition. In contrast, in a case of abortion of an African elephant, the second peak of ir-PRL was not observed, and the levels remained low for about four months until parturition. The weight of the fetus delivered at the 17th month of gestation was 23.5 kg, which was quite small compared with normal fetuses in previous reports. Ir-PRL was detected in placental homogenates, and immunolocalization was observed in trophoblasts in both the African and Asian elephants, indicating that the placenta is the source of ir-PRL during pregnancy in elephants. The present results clearly demonstrated that circulating ir-PRL shows a biphasic pattern during normal pregnancy and that the placenta appears to be an important source of circulating ir-PRL during pregnancy in both African and Asian elephants.     

Seasonal changes of the mineral contents in the rumen of wild Yeso sika deer (Cervus nippon yesoensis)

The rumen contents were collected from 36 wild Yeso sika deer (Cervus nippon yesoensis) captured by deer culling or by hunting in the spring, summer, autumn and winter in Hokkaido, Japan. Botanical classification was conducted, and the contents of mineral (calcium (Ca), phosphorus (P), potassium (K), sodium (Na), iron (Fe), copper (Cu) and zinc (Zn)) were measured. The animals were captured around pastures or fallow field areas in the Kushiro area. The rumen contents consisted of grasses and Sasa sp. leaves regardless of the season. Leaves and bark were ingested in the spring, autumn and winter. The macro‐mineral contents in the rumen showed seasonal changes. In the summer, the Ca, K and P contents were high, and the Na content was low. There were no seasonal changes in the Fe content. The P, Na and Fe contents were higher than the animals' requirements. In a future survey, it is needed to determine the mineral contents of the food ingested by wild Yeso sika deer.     

Identification of heat shock protein 70 in canine reticulocytes and mature erythrocytes

In the present study, we demonstrated that heat shock protein 70 (Hsp70) was present in both canine reticulocytes and mature erythrocytes, and that the canine Hsp70 in reticulocytes was decreased along with the maturation of the cells into erythrocytes. These results suggest that the Hsp70 in canine reticulocytes might act as a chaperone to remove unnecessary proteins during reticulocyte maturation. We also demonstrated that Hsp70 was present in exosomes from reticulocytes during their maturation in in vitro culture. Furthermore, the concentration of Hsp70 in reticulocyte membranes was increased in proportion to an increase of the protein in exosomes until 48 hours after the incubation of reticulocytes in vitro. At 96 hours of the incubation, however, only a trace amount of Hsp70 was detected in the membrane, while a large amount of the protein was present in the exosomes. These results suggest that Hsp70 in canine reticulocytes might play an important role for exosome formation in reticulocytes, resulting in the maturation of the cells.     

Heinz body hemolytic anemia with eccentrocytosis from ingestion of Chinese chive (Allium tuberosum) and garlic (Allium sativum) in a dog

A 4-year-old, intact male miniature schnauzer was presented with anorexia. The dog had ingested some Chinese steamed dumplings 2 days before, which contained Chinese chive (Allium tuberosum) and garlic (Allium sativum). Hematological examinations revealed severe Heinz body hemolytic anemia with eccentrocytosis and an increased concentration of methemoglobin, which was thought to result from oxidative damage to erythrocytes by constituents in these Allium plants. In this case, eccentrocytosis was a hallmark finding and could be detected easily, suggesting that this hematological abnormality is useful in diagnosing Allium plant-induced hemolysis.     

Cloning and expression of canine interferon-alpha genes in Escherichia coli

We cloned five new subtypes of cDNA encoding canine interferon-alpha (CaIFN-alpha) from a canine epithelial cell line. CaIFN-alphas were divided into two groups by amino acid sequences and a molecular phylogenic tree. Two subtypes of them were expressed in Escherichia coli, and IFN proteins were purified. Recombinant CaIFN-alphas were highly species-specific and showed antiviral activity against Vesicular stomatitis New Jersey virus and canine adenovirus-1 , but not against canine herpesvirus-1.     

Relation between reticulocyte count and characteristics of erythrocyte 5'-nucleotidase in dogs,cats, cattle and humans

To examine substrate specificity and susceptibility to lead, erythrocyte 5'-nucleotidase was measured in dogs, cats, cattle and humans, and its relationship to the reticulocyte count in these species was determined. The reticulocyte count in dogs was similar to that in humans, but the count in cats was higher than that in humans. Reticulocytes were not observed in cattle. The activities of canine erythrocyte 5'-nucleotidase measured using cytidine and uridine 5'-monophosphates, which are preferentially catalyzed by one of the human pyrimidine 5'-nucleotidase isozymes (P5N-I), were similar to those of the human enzyme. The canine enzyme preferentially catalyzed thymidine 3'-monophosphate, which is catalyzed only by human P5N-II, more strongly than the human enzyme. This suggests that canine erythrocytes have two isozymes similar to human P5N-I and P5N-II, and a higher P5N-II-like activity than human erythrocytes. Feline erythrocytes had the highest level of P5N-I-like activity among the species examined, and the bovine enzymic activities including those of P5N-I and II were the lowest among these species. According to these observations, the reticulocyte count was approximately proportional to the P5N-I-like activity in these species. Therefore, the P5N-I-like activity may be involved in the morphological maturation of mammalian erythrocytes. The canine and feline erythrocytes had markedly high activity and preferentially catalyzed purine 5'-monophosphate suggesting the presence of a purine-specific 5'-nucleotidase as in human erythrocytes. In addition, the canine and feline P5N-I-like activity showed less susceptibility to lead than the human P5N-I. This may be a reason why there are few case reports of lead-induced anemia in dogs and cats.     

Clinical and clinico-pathologic characteristics of Shiba dogs with a deficiency of lysosomal acid beta-galactosidase: a canine model of human GM1 gangliosidosis

The present study was conducted to determine the clinical and clinico-pathologic characteristics of Shiba dogs with GM1 gangliosidosis, which is due to an autosomal recessively inherited deficiency of lysosomal acid beta-galactosidase activity. Clinical and clinico-pathological features were investigated in 10 homozygous Shiba dogs with GM1 gangliosidosis. The age at onset was 5 to 6 months and the dogs manifested progressive neurologic signs including loss of balance, intermittent lameness, ataxia, dysmetria and intention tremor of the head. The dogs were unable to stand by 10 months of age due to a progression of ataxia and spasticity in all limbs. Corneal clouding, a visual defect, generalized muscle rigospasticity, emotional disorder and a tendency to be lethargic were observed at 9 to 12 months. The dogs became lethargic from 13 months of age. The survival period seemed to be 14 to 15 months. As a clinico-pathologic feature, lymphocytes with abnormally large vacuoles were observed in peripheral blood (30 to 50% of total lymphocytes) through the lifetime of the dogs. The clinical and clinico-pathologic characteristics of this animal model are useful for not only the development and testing of potential methods of therapy, but also the diagnosis of affected homozygous Shiba dogs in veterinary clinics.     

An enzyme-linked immunosorbent assay with the recombinant merozoite protein as antigen for detection of antibodies to Eimeria necatrix

A cDNA library was constructed with Eimeria necatrix merozoite mRNA and immunologically screened by chicken sera against this parasite. One of the positive clones containing an insert of 879 nucleotides, pNP19, showed similarity to part of a published gene expressed in E. tenella merozoite by the homology search system. The inserted DNA was subcloned into baculovirus, and a 35-kD protein was expressed, purified, and used for the antigen in enzyme-linked immunosorbent assay (ELISA). Antibodies from the chickens vaccinated with the E. necatrix attenuated strain, Nn-P125, were detected from 14 days after vaccination by ELISA. The mean absorbance increased rapidly to a peak around 21 days after vaccination; thereafter, it began to decline. Even though some of the vaccinated chickens showed very low levels of antibody response to the recombinant protein 56 days after vaccination, they were protected against challenge with virulent strain of E. necatrix. The mean absorbances in sera from both vaccinated and nonvaccinated chickens highly increased 14 days after challenge. On the other hand, the antibody was not detected in ELISA when chickens were exposed to other Eimeria species such as E. tenella, E. acervulina, and E. maxima. These results demonstrate that this recombinant protein is suitable for detecting the specific antibody in chickens infected with both attenuated and virulent strains of E. necatrix.     

Increased concentration of GM1-ganglioside in cerebrospinal fluid in dogs with GM1- and GM2-gangliosidoses and its clinical application for diagnosis.

GM1- and GM2-gangliosidoses are lethal lysosomal diseases that are caused by a defect of acid hydrolases, resulting in the intralysosomal accumulation of the specific physiological substrates, GM1- and GM2-gangliosides, respectively. In the present study a method for the diagnosis of canine GM1-gangliosidosis was established using canine cerebrospinal fluid (CSF). The concentration of GM1-ganglioside in CSF was determined by thin-layer chromatography-enzyme immunostaining using biotin-conjugated cholera toxin B, which specifically binds with GM1-ganglioside. The concentration of CSF GM1-ganglioside was increased in Shiba dogs with GM1-gangliosidosis, and the increased level was approximately proportional to the age of the dogs. The concentration was high in the affected dog even at 5 months of age, when Shiba dogs with GM1-gangliosidosis first manifest neurologic signs. In addition, the concentration of CSF GM1-ganglioside in a dog with the GM2-gangliosidosis 0 variant (Sandhoff disease) was also 7 times the normal level. From these results it was concluded that this laboratory technique enables a definitive and early diagnosis of canine GM1-gangliosidosis even if tissues and organs cannot be obtained. However, because GM1-ganglioside can also be elevated in cases of GM2-gangliosidosis, it is necessary to assay for specific enzyme deficiencies to definitively separate GM1- from GM2-gangliosidosis.