Rapid detection of GM1 ganglioside in cerebrospinal fluid in dogs with GM1 gangliosidosis using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

The concentration of GM1 (monosialotetrahexosyl ganglioside) in cerebrospinal fluid (CSF) is markedly increased in dogs with GM1 gangliosidosis due to GM1 accumulation in the central nervous system and leakage to the CSF. The present study established a rapid and simple method for detection of accumulated GM1 in the CSF in dogs with GM1 gangliosidosis using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MS) and discusses the usefulness of this method for the rapid diagnosis and/or high-risk screening of this disease in domestic animals. Cerebrospinal fluid was collected from normal dogs and 4- to 11-month-old Shiba dogs with GM1 gangliosidosis. The MALDI TOF MS analysis was carried out in combination with a special sample plate and a simple desalting step on the plate. Specific signs of GM1 could be detected in the standard GM1 solutions at concentrations of 50 nmol/l or more. The signs were also clearly detected in CSF (131-618 nmol/l) in affected dogs, but not in normal canine CSF (12 ± 5 nmol/l, mean ± standard deviation). The results demonstrated that MALDI TOF MS can detect GM1 accumulated in canine CSF even in the early stage of the disease. In conclusion, the rapid detection of increased CSF GM1 using MALDI TOF MS is a useful method for diagnosis and/or screening for canine GM1 gangliosidosis.

Babesia gibsoni: detection in blood smears and formalin-fixed, paraffin-embedded tissues using deoxyribonucleic acid in situ hybridization analysis.

In this study, we attempted to detect Babesia gibsoni in blood smears and formalin-fixed, paraffin-embedded tissues obtained from B. gibsoni-infected dogs using in situ hybridization. Using a digoxigenin-conjugated deoxyribonucleic acid (DNA) probe, both intraerythrocytic and exoerythrocytic parasites in the culture could be specifically stained in blood smears fixed with 4% phosphate-buffered paraformaldehyde. This indicated that genomic DNA extracted from the parasites could be detected using in situ hybridization. Moreover, the parasite could be specifically stained in paraffin-embedded spleen, lymph node, and kidney sections using in situ hybridization. Infected erythrocytes in blood vessels in the spleen and kidney, hemosiderin-laden macrophages in the spleen, and phagocytized erythrocytes, which seemed to be infected with the parasites, in lymph nodes were also specifically stained. This suggests that in situ hybridization can be utilized to investigate both the life cycle of B. gibsoni and the pathological condition of canine babesiosis.

Reduced transcript levels of the heat shock protein 70 gene in diminazene aceturate-resistant Babesia gibsoni variants under low concentrations of diminazene aceturate.

In our previous report, we developed a diminazene aceturate (DA)-resistant Babesia gibsoni strain that was maintained in culture with 200 ng/ml DA. While developing this strain, we also obtained DA-resistant B. gibsoni variants, which were maintained in culture with DA from 1 to 175 ng/ml for more than 8 weeks. Because heat shock protein 70 (Hsp70) seems to play important roles in adaptation to a stress environment in protozoan parasites, in the present study, we examined the copy number of B. gibsoni Hsp70 (BgHsp70) transcripts of those DA-resistant variants using quantitative real-time reverse transcription-polymerase chain reaction. We found that when wild-type B. gibsoni was exposed to 1 ng/ml DA, the level of BgHsp70 transcripts was decreased at day 14. The copy number of BgHsp70 transcripts in the DA-resistant variant cultured with 1 ng/ml DA was significantly lower than in wild-type B. gibsoni, while those in DA-resistant variants increased with escalating doses of DA from 1 to 75 ng/ml, although they were lower than in wild-type B. gibsoni. However, those in DA-resistant variants cultured with >125 ng/ml DA were almost the same as wild-type B. gibsoni. These results indicated that the transcript levels of the BgHsp70 gene might be reduced when the parasites are exposed to a low concentration of DA, and then might recover to the normal level after achieving resistance against DA. We expect that further study of the function of BgHsp70 will elucidate the mechanism of drug resistance against DA in B. gibsoni.

Development and characterization of a strain of Babesia gibsoni resistant to diminazene aceturate in vitro.

We attempted to develop a strain of Babesia gibsoni resistant to diminazene aceturate (DA), an anti-babesial drug, in vitro. Since the DA-sensitive B. gibsoni strain could survive and proliferate in culture medium containing 1 ng/m l DA, the concentration of DA was gradually increased from 1 to 200 ng/ml. The results showed that the parasites could survive and proliferate in the medium containing 200 ng/m l DA, which was much higher than the 50% inhibitory concentration (IC(50)) of DA for B. gibsoni. Subsequently, these parasites were removed from erythrocytes and exposed directly to 200 ng/ml DA. They were able to survive and invade fresh erythrocytes, though the DA-sensitive B. gibsoni strain did not survive. Based on these results, the parasites cultured within 200 ng/ml DA were determined to be a DA-resistant B. gibsoni strain. In addition, the IC(50) levels of clindamycin, doxycycline and pentamidine for the DA-resistant B. gibsoni strain were determined. The IC(50) levels of clindamycin, doxycycline and pentamidine for the DA-resistant strain were higher than those for the DA-sensitive strain. The IC(50) of pentamidine for the resistant strain was much greater than that for the DA-sensitive strain. These results indicated that the DA-resistant B. gibsoni strain could have resistance not only to DA, but also to other anti-babesial drugs. In conclusion, we successfully developed a DA-resistant B. gibsoni strain in vitro.

Evaluation of efficacy of bruceine A, a natural quassinoid compound extracted from a medicinal plant, Brucea javanica, for canine babesiosis.

Bruceine A, a natural quassinoid compound extracted from the dried fruits of Brucea javanica (L.) Merr., was evaluated for its antibabesial activity in vitro and in vivo. Bruceine A inhibited the in vitro growth of Babesia gibsoni in canine erythrocytes at lower concentration compared with the standard antibabesial drug diminazene aceturate and killed the parasites within 24 hr at a concentration of 25 nM. Oral administration of bruceine A at a dosage of 6.4 mg/kg/day for 5 days resulted in no clinical findings in a dog with normal ranges of hematological and biochemical values in the blood. Three dogs were infected with B. gibsoni and two of them were treated with bruceine A at a dosage of 6.4 mg/kg/day for 6 days from day 5 post-infection. An untreated dog developed typical acute babesiosis symptoms including severe anemia, high fever, and complete loss of appetite and movement. However, the two bruceine A-treated dogs maintained their healthy conditions throughout the experimental period of 4 weeks although complete elimination of parasites from the peripheral blood was not achieved and decreases in the packed cell volume and the erythrocyte and platelet counts were observed. Since natural quassinoid compounds have been used as traditional medicines for the treatment of various ailments including cancer and malaria, the present results suggest that bruceine A or other related compounds are potential candidates for the treatment of canine babesiosis.

Effects and mechanisms of action of ionophorous antibiotics valinomycin and salinomycin-Na on Babesia gibsoni in vitro.

Valinomycin and salinomycin-Na, 2 ionophorous antibiotics, exhibited in vitro antibabesial activities against Babesia gibsoni that infected normal canine erythrocytes containing low potassium (LK) and high sodium concentrations, i.e., LK erythrocytes, which completely lack Na,K-ATPase activity. The level of parasitemia of B. gibsoni was significantly decreased when the parasites were incubated in culture medium containing either 10(-1) ng/ml valinomycin or 10(2) ng/ml salinomycin-Na for 24 hr. Four-hour incubation in the culture medium containing 5 µg/ml salinomycin-Na led to the destruction of most parasites. In contrast, when the parasites infected canine erythrocytes containing high potassium (HK) and low sodium concentrations, i.e., HK erythrocytes, the in vitro antibabesial activities of both ionophorous antibiotics seemed to be weakened, apparently due to the protection by the host cells. Therefore, differential influences of ionophorous antibiotics on LK and HK erythrocytes were observed. In LK erythrocytes, the intracellular concentrations of potassium, sodium, and adenosine triphosphate (ATP) were not modified, and hemolysis was not observed after incubation in the medium containing each ionophorous antibiotic. These results suggested that these ionophorous antibiotics did not affect cells without Na,K-ATPase, and directly affected B. gibsoni. In HK erythrocytes, the ionophorous antibiotics increased the intracellular sodium concentration, and decreased the intracellular potassium and ATP concentrations, causing obvious hemolysis. Additionally, the decrease of the intracellular ATP concentration and the hemolysis in HK erythrocytes caused by valinomycin disappeared when the activity of Na,K-ATPase was inhibited by ouabain. These results indicate that modification of the intracellular cation concentrations by the ionophorous antibiotics led to the activation of Na,K-ATPase and increased consumption of intracellular ATP, and that the depletion of intracellular ATP resulted in hemolysis in HK erythrocytes. Moreover, the antibabesial activity of valinomycin disappeared when B. gibsoni in LK erythrocytes were incubated in culture media containing high potassium concentrations. This showed that the intracellular cation concentration in the parasites was not modified in those media and would remain the same.

Retrospective diagnosis of feline GM2 gangliosidosis variant 0 (Sandhoff-like disease) in Japan: possible spread of the mutant allele in the Japanese domestic cat population.

GM2 gangliosidosis variant 0 (human Sandhoff disease) is a lysosomal storage disease caused by simultaneous deficiencies of acid beta-hexosaminidase (Hex) A and Hex B due to an abnormality of beta-subunit, a common component in these enzyme molecules, which is coded by the HEXB gene. In the present study, a retrospective diagnosis was performed in 2 previous suspected cases of feline Sandhoff-like disease using a DNA test to detect the causative mutation identified previously in 4 cats in 2 other families of Japanese domestic cats. Enzymic analysis was also performed using stored leukocytes and plasma collected from the subject families in order to investigate the usefulness of enzymic diagnosis and genotyping of carriers. The DNA test suggested that the 2 cases were homozygous recessive for the mutation. Consequently, 6 cats homozygous for the same mutation have been found in 4 separate locations of Japan, suggesting that this mutant allele may be spread widely in the Japanese domestic cat populations. In enzymic analysis, Hex A and Hex B activities in leukocytes and plasma measured using 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide as a substrate were negligible in affected cats, compared with those in normal and carrier cats. However, there was a wide overlap in enzyme activity between normal and carrier cats. Therefore, it was concluded that enzymic analysis is useful for diagnosis of affected cats, but is not acceptable for genotyping of carriers.

Flow cytometry to evaluate the level of Babesia gibsoni parasitemia in vivo and in vitro by using the fluorescent nucleic acid stain SYTO16.

In the present study, we employed flow cytometry to evaluate the level of parasitemia of Babesia gibsoni infecting canine erythrocytes in vivo and in vitro by using fluorescent nucleic acid staining. Peripheral blood samples from a B. gibsoni-infected dog and cultured B. gibsoni parasitizing in canine erythrocytes were stained with a membrane-permeable fluorescent nucleic acid stain, SYTO16. In this study, we utilized normal canine erythrocytes (LK erythrocytes) and canine erythrocytes containing high concentrations of potassium, reduced glutathione, and some free amino acids (HK erythrocytes) as host cells for culture. Parasitized cells in vive were discriminated completely from unparasitized cells and a correlation (r = 0.998) between the percentage of SYTO16-positive cells and parasitemia in vivo was observed. On the other hand, erythrocytes in vitro could not be divided clearly into parasitized and unparasitized cells. However, when LK erythrocytes were used as host cells, the percentage of SYTO16-positive cells was almost the same as, and was well correlated (r = 0.932) with, the level of parasitemia. When HK erythrocytes were used as host cells, the percentage of SYTO16-positive cells was almost half of, but was correlated (r = 0.982) with, the level of parasitemia. Therefore, we attempted to observe the changes in the percentage of parasitized cells after treatment with antiprotozoal drug or mitochondria inhibitors by using flow cytometry. The changes in the percentage of SYTO16-positive cells corresponded well with the changes of the level of parasitemia when the parasites in HK erythrocytes were cultured with each compound. The present results suggest that flow cytometric detection using SYTO16 is a rapid and reliable method for monitoring parasitemia both in vive and in vitro.

Heat shock response of Babesia gibsoni heat shock protein 70.

mRNA and protein expression profiles for heat shock protein 70 (Hsp70) of Babesia gibsoni (BgHsp70) exposed to either high or low temperatures, were examined by quantitative real-time reverse transcription-polymerase chain reaction and Western blotting. In the present study, a commercially available anti-human Hsp70 antibody that could recognize recombinant BgHsp70 was used. BgHsp70 was detected in the parasites cultured under standard conditions at 37 C by Western blotting and immunostaining of thin smears of infected erythrocytes. These results suggested that BgHsp70 was expressed constitutively at the erythrocyte stage. BgHsp70 levels were elevated when the parasites were incubated at 42 C for 1 hr. In contrast, its mRNA amount was decreased and its protein amount was unchanged when the parasites were incubated at 32 C for 1 hr. Moreover, the level of parasitemia of B. gibsoni incubated at either 42 C or 32 C was almost the same as that at 37 C. These results indicated that the exposure of B. gibsoni to elevated temperatures might result in increased expression of BgHsp70 and that the exposure of the intraerythrocytic parasites to decreased temperatures might not induce the overexpression of BgHsp70.

Molecular screening of canine GM1 gangliosidosis using blood smear specimens after prolonged storage: detection of carriers among shiba dogs in northern Japan.

Molecular screening of GM1 gangliosidosis in Shiba dogs was carried out in northern Japan using blood smear specimens after prolonged storage. Of 125 specimens obtained from 3 veterinary teaching hospitals for this screening, 68 specimens (54%) were adequate for direct amplification in a polymerase chain reaction (PCR)-based DNA test, and the percentage of adequacy was different at each hospital (34%, 73%, and 100%), suggesting that the amount of blood on the smear and the storage condition of specimens may affect adequacy. Of the 68 dogs examined, 2 dogs (2.9%) were heterozygous carriers for this disease and the other dogs were all genotypically normal. The results suggest blood smear specimens can be useful for PCR testing after prolonged storage provided specimens contain a generous amount of blood and have been adequately stored. The study also suggests that GM1 gangliosidosis may be widely prevalent in the Shiba dog population in northern Japan.

Screening of Indonesian medicinal plant extracts for antibabesial activity and isolation of new quassinoids from Brucea javanica.

Boiled extracts derived from 28 Indonesian medicinal plants were screened for their antibabesial activity against Babesia gibsoni in vitro. Of these extracts, the fruit of Brucea javanica was the most active in inhibiting parasite growth at a concentration of 10 microg/mL. Bioassay-guided fractionation of the fruit extract of Br. javanica led to the isolation of two new quassinoids, bruceantinol B and bruceine J, and the structures of these compounds were elucidated on the basis of their spectroscopic data and by chemical transformation to known compounds. In addition, the known quassinoids bruceines A-D, bruceantinol, and yadanziolide A were isolated. Antibabesial activities were also examined in vitro, and bruceine A and bruceantinol were shown to be more potent than diminazene aceturate, a drug (IC50 = 103 ng/mL) used clinically against B. gibsoni, with IC50 values of 4 and 12 ng/mL, respectively.

Clinico-pathological findings in peripartum dairy cows fed anion salts lowering the dietary cation-anion difference: involvement of serum inorganic phosphorus, chloride and plasma estrogen concentrations in milk fever.

In our previous study, it was demonstrated that the administration of anion salts, which slightly lower the dietary cation-anion difference (DCAD), in the prepartum period is safe and effective for preventing milk fever in multiparous cows. In the present study, several clinico-pathological constituents in serum and urine, which might be related to milk fever, were analyzed using stored samples from the previous study to identify clinico-pathological parameters for easily evaluating the efficacy of lowering DCAD and to further investigate the mechanism by which lowering DCAD prevents milk fever. Among the parameters analyzed in the present study, inorganic phosphorus (iP) was involved in milk fever because the serum concentration and urinary excretion of iP were significantly higher in the group of primiparous cows (heifer group), which did not develop hypocalcemia, than those in other groups of multiparous cows. Serum chloride concentrations in the heifer group and the group of multiparous cows fed anion salts (anion group) tended to remain higher than those in other control groups of multiparous cows suggesting that serum chloride concentration may be utilized for evaluating the status of metabolic acidosis and the efficacy of lowerng DCAD in dairy cows fed anion salts. In addition, plasma estradiol-17beta concentration in the heifer group tended to be lower at parturition compared with that in other multiparous groups suggesting that estrogen known as a potent inhibitor of bone resorption may be involved in developing milk fever.

Suppressive effect of culture supernatant of erythrocytes and serum from dogs infected with Babesia gibsoni on the morphological maturation of canine reticulocytes in vitro.

The present study evaluated the effects of infected culture supernatant of erythrocytes, fractionation of culture supernatant and serum from dogs infected with Babesia gibsoni (B. gibsoni) on the maturation of canine reticulocytes in vitro. The SDS-PAGE demonstrated that significantly broader bands were generated by both the infected culture supernatant of erythrocytes and the serum from dogs chronically infected with B. gibsoni. The culture supernatant of erythrocytes infected with B. gibsoni strongly suppressed the maturation of reticulocytes. Prior studies showed that chronically infected serum had inhibitory effects on both the maturation of reticulocytes and the canine pyrimidine 5'-nucleotidase subclass I and purine-specific 5'-nucleotidase activity. In addition, serum free infected culture supernatant of erythrocytes had an inhibitory effect on the morphological maturation of reticulocytes. These results suggest that infected serum and culture supernatant of erythrocytes might accumulate excess proteins and/or metabolites as a result of the inhibited maturation of reticulocytes and decreased activity of erythrocyte 5'-nucleotidase. Furthermore, the fractions observed at >150 kDa- and 150-70 kDa- in the infected culture supernatant and serum retarded the maturation of canine reticulocytes in vitro. The results obtained from the in vitro examinations, in the present study, suggested that B. gibsoni itself and/or its metabolites might release certain proteins in the infected culture supernatant and serum from infected dogs and as a result delay morphological maturation of canine reticulocytes.

Anti-babesial compounds from Curcuma xanthorrhiza.

Anti-babesial ingredients, (12R)- and (12S)-12,13-dihydro-12,13-dihydroxyxanthorrhizols, were isolated from Curcuma xanthorrhiza. The structures were established by the extensive NMR techniques. The assignments of (1)H NMR data of (12R)-12,13-dihydro-12,13-dihydroxyxanthorrhizol was revised, and (12S)-12,13-dihydro-12,13-dihydroxyxanthorrhizol was isolated as a pure form for the first time. The IC(50) of the active compounds were compared with that of commercial drug, diminazene aceturate (Ganaseg). IC(50) values of Ganaseg, (12R)-, and (12S)-12,13-dihydro-12,13-dihydroxyxanthorrhizols were 0.6 microg mL(-1), 8.3 microg mL(-1) and 11.6 microg mL(-1), respectively.

Biomarkers for the activation of calcium metabolism in dairy cows: elevation of tartrate-resistant acid phosphatase activity by lowering dietary cation-anion difference is associated with the prevention of milk fever.

In our previous study, it was demonstrated that the administration of anion salts, which slightly lower the dietary cation-anion difference (DCAD), in the prepartum period is safe and effective for preventing milk fever in multiparous cows. In the present study, several biomarkers, which might show activation of Ca metabolism, were analyzed using stored samples in the previous study to investigate the mechanism of the preventive effect on milk fever by lowering DCAD. Changes in bone-specific alkaline phosphatase activity, osteocalcin and insulin-like growth factor I concentrations in serum were almost the same among the three groups of multiparous cows with or without the oral administration of anion salts, while the levels of these serum biomarkers in the group of primiparous cows (heifer group) were much higher compared with those in the three multiparous groups throughout the experimental period. Urinary deoxypyridinoline excretion was not a useful biomarker for dairy cows because it hardly changed during the peripartum period in all groups. However, serum tartrate-resistant acid phosphatase (TRAP) activity, which is known as a biomarker of osteoclast activity, was well associated with the administration of anion salts lowering DCAD because among the three multiparous groups, only the group of multiparous cows fed the anion salts (anion group) showed an increased level, which rose to the level in the heifer group, and was markedly higher than those in the other control groups of multiparous cows. The increased activity of serum TRAP in the anion group suggested that Ca in the plasma pool was mobilized smoothly from bone-bound Ca via mature osteoclasts at parturition, which might be due to prior activation under mild acidosis induced by slightly lowering DCAD. Therefore, TRAP was the best biomarker to monitor the activation of Ca metabolism in dairy cows fed anion salts.

Preventive effect of mildly altering dietary cation-anion difference on milk fever in dairy cows.

In the present study, we examined whether mildly altering dietary cation-anion difference (DCAD) contributes to the prevention of milk fever in dairy cows. Thirty multiparous cows and ten primiparous cows (heifer group) were used in this study and the multiparous cows were randomly divided into three groups of ten animals each (anion, non-anion and control groups). The cows in the anion group were given supplemental salts that slightly lowered DCAD. These salts consisted of 115 g of CaCO3, 42 g of CaHPO4, 65 g of MgSO4 x 7 H2O and 80 g of CaCl2 x 2 H2O as a daily dose for each cow, using a catheter from 21 days before the expected date of parturition until parturition. The cows in the non-anion group were given only the same Ca, Mg and ip supplement but no sulfate and chloride salts as that in the anion group. The cows in the control and heifer groups were not given any additional supplement. The incidence of hypocalcemia in the anion group decreased to approximately half of those in the non-anion and control groups, while the heifer group did not develop hypocalcemia at all. In addition, the number of days spent for the treatment of hypocalcemia and the number of drug bottles (calcium borogluconate solution) used for the treatment decreased to less than half in the anion group compared with those in the non-anion and control groups. At parturition, the serum Ca concentration in the control (6.2 +/- 1.9 mg/dl, mean +/- standard deviation) and non-anion groups (6.4 +/- 1.7 mg/dl) were significantly lower than that in the heifer group (8.3 +/- 0.4 mg/dl), and the level in the anion group was intermediate (7.3 +/- 1.3 mg/dl). The change in ionized Ca concentration was almost the same as that in serum Ca concentration, but only the concentration in the anion group tended to increase slightly from a week before parturition and was significantly higher than that in all other groups three days before parturition. Urinary pH in the anion group was maintained at a mildly acidic level (6.8-7.0) for the last two weeks before parturition, compared with those in the control (7.3-7.5) and non-anion groups (7.9-8.1), and similar to that in the heifer group (6.3-7.3). The urinary Ca excretion was the highest in the anion group among all groups during the prepartum period. There were no specific changes in the excretion of parathyroid hormone and 1,25-dihydroxyvitamin D in all groups of multiparous cows while the levels of these hormones remained low in the heifer group throughout the experimental period. The data in the present study indicates that the administration of anion salts that slightly lowered DCAD in the preparum period was effective for preventing milk fever in multiparous cows. Safe and mild metabolic acidosis induced by the anion salts could be evaluated by urinary pH (6.8-7.0), and might increase the responsiveness to Ca requirement at parturition through some complex mechanisms unrelated to the excretion of Ca-related hormones. In addition, it was clarified that primiparous cows have a high potential to respond to sudden Ca demand unrelated to hormone excretion, and their Ca metabolism was in some respects similar to that in multiparous cows fed anion salts. Therefore, manipulating mildly DCAD is expected to be an effective, safe and natural method for preventing milk fever in dairy cows.

Comparison and phylogenetic analysis of the heat shock protein 70 gene of Babesia parasites from dogs.

The heat shock protein 70 (hsp70) genes of Babesia gibsoni, B. canis canis, B. canis vogeli, and B. canis rossi isolated from infected dogs were cloned by polymerase chain reaction (PCR) and sequenced. In the nucleotide sequence and the predicted amino acid sequence of the gene, the parasites were very similar to each other. The nucleotide sequences of the hsp70 gene had more variety than those of 18S nuclear subunit ribosomal DNA (18S rDNA). A phylogenetic analysis of these sequences and comparisons with sequences from other Babesia and Theileria species revealed that all canine babesial isolates analyzed in the present study were closely related to each other and formed one cluster. Additionally, a phylogenetic analysis of Babesia and Theileria species showed that these parasites could be divided into three groups: group A including canine babesial isolates, B. divergens, B. odocoilei, B. bovis, B. caballi, and B. ovis; group B including Theileria annulata, T. orientalis, and T. cervi; and group C including B. microti and B. rodhaini. These results suggested that a phylogenetic analysis of the hsp70 gene sequence might be helpful in classifying Babesia and Theileria species, and that canine babesial isolates might be closely related to each other, indicating their evolution from the same ancestry.

Clinical and molecular analysis of GM2 gangliosidosis in two apparent littermate kittens of the Japanese domestic cat.

This case report documents clinical and molecular findings in two littermate kittens of the Japanese domestic cat with GM2 gangliosidosis variant 0. Analysis included detailed physical, magnetic resonance imaging, biochemical, pathological and genetic examinations. At first, these littermate kittens showed typical cerebellar signs at approximately 2 months of age. About 2 months later, they progressively showed other neurological signs and subsequently died at about 7 months of age. Magnetic resonance imaging just before the death showed an enlarged ventricular system, T1 hyperintensity in the internal capsule, and T2 hyperintensity in the white matter of the whole brain. Histological findings suggested a type of lysosomal storage disease. Biochemical studies demonstrated that the kittens were affected with GM2 gangliosidosis variant 0, and a DNA assay finally demonstrated that these animals were homozygous for the mutation, which the authors had identified in a different family of the Japanese domestic cat. The findings in the present cases provide useful information about GM2 gangliosidosis variant 0 in Japanese domestic cats.

Cerebrospinal fluid biomarkers showing neurodegeneration in dogs with GM1 gangliosidosis: possible use for assessment of a therapeutic regimen.

The present study investigated cerebrospinal fluid (CSF) biomarkers for estimating degeneration of the central nervous system (CNS) in experimental dogs with GM1 gangliosidosis and preliminarily evaluated the efficacy of long-term glucocorticoid therapy for GM1 gangliosidosis using the biomarkers identified here. GM1 gangliosidosis, a lysosomal storage disease that affects the brain and multiple systemic organs, is due to an autosomal recessively inherited deficiency of acid beta-galactosidase activity. Pathogenesis of GM1 gangliosidosis may include neuronal apoptosis and abnormal axoplasmic transport and inflammatory response, which are perhaps consequent to massive neuronal storage of GM1 ganglioside. In the present study, we assessed some possible CSF biomarkers, such as GM1 ganglioside, aspartate aminotransferase (AST), lactate dehydrogenase (LDH), neuron-specific enolase (NSE) and myelin basic protein (MBP). Periodic studies demonstrated that GM1 ganglioside concentration, activities of AST and LDH, and concentrations of NSE and MBP in CSF were significantly higher in dogs with GM1 gangliosidosis than those in control dogs, and their changes were well related with the months of age and clinical course. In conclusion, GM1 ganglioside, AST, LDH, NSE and MBP could be utilized as CSF biomarkers showing CNS degeneration in dogs with GM1 gangliosidosis to evaluate the efficacy of novel therapies proposed for this disease. In addition, we preliminarily treated an affected dog with long-term oral administration of prednisolone and evaluated the efficacy of this therapeutic trial using CSF biomarkers determined in the present study. However, this treatment did not change either the clinical course or the CSF biomarkers of the affected dog, suggesting that glucocorticoid therapy would not be effective for treating GM1 gangliosidosis.

Use of amnion and placenta in neonatal screening for canine GM1-gangliosidosis and the risk of diagnostic misclassifications.

BACKGROUND: A closed breeding colony of Shiba dogs with GM1-gangliosidosis is maintained at Hokkaido University (Sapporo, Japan). Neonatal genotyping is essential to control the breeding colony genetically as an animal model for the human disease. OBJECTIVES: The purpose of the present study was to determine the utility of amnion and placenta in the neonatal screening or diagnosis for canine GM1-gangliosidosis. METHODS: Twenty neonatal Shiba dogs of a pedigree with GM1- gangliosidosis were differentiated into 3 genotypes--normal, heterozygous, and affected dogs--by using a previously reported DNA mutation assay. Acid beta-galactosidase activity was measured in amnion and placenta and compared among the 3 genotypes. RESULTS: The level of beta-galactosidase activity in the amnion of affected dogs was negligible and <2% of the mean activity in normal dogs; there was no significant difference among the 3 genotypes. In placenta, beta-galactosidase activity was significantly different among all the genotypes; however, there was wide overlap in enzyme activity between normal and heterozygous dogs. The level of activity in affected dogs was relatively high and >10% of the mean activity in normal dogs. The DNA mutation assay gave correct information about genotype with genomic DNA extracted from amnion but ambiguous information with DNA from placenta. CONCLUSIONS: Amnion and placenta were not useful as enzyme sources in neonatal screening in canine GM1-gangliosidosis because of the risk of misdiagnosis. DNA from amnion is applicable as a template for genotyping, whereas placenta should not be used because canine placenta contains maternal cells.