Mitochondrial DNA analysis of Nepalese domestic dwarf cattle Lulu*

Dwarf Lulu cattle, the only Bos Taurus type of cattle in Nepal, are raised under severe environments in the mountainous zone of that country. In the present study, the body measurement traits, cytogenetic and molecular genetic characteristics of the Lulu cattle are investigated. Blood samples were collected from 31 animals in four villages (altitudes 2590–3550 m) in the southern part of Mustang. The Lulu cattle had a normal karyotype with 2n = 60, XY or XX. Only one male examined had a large submetacentric X‐chromosome and a small submetacentric taurine type Y‐chromosome. The mitochodrial DNA (mtDNA) genotypes were analyzed by PCR mediated restriction fragment length polymorphisms, displacement (D)‐loop region PCR mediated single strand conformation polymorphisms, and D‐loop region sequences. Many base substitutions were found in the D‐loop region, suggesting that the Lulu cattle originated from at least 10 maternal lines. Three types of mtDNA from these cattle were found, the Bos taurus type (n = 23), the Bos indicus type (n = 6), and the Bos grunniens type (n = 2). In the village at the lowest altitude, four of the five cows were of the Bos indicus type. These results indicated that mtDNA types of the Lulu cattle mostly belong to Bos taurus, but have been hybridized with Bos indicus cattle in lower‐elevation regions in their maternal lineage.     

Pathological analysis of batch safety testing of veterinary vaccines using small laboratory animals

Batch safety tests (BSTs) of veterinary vaccines are conducted using small laboratory animals to assure the safety of vaccines according to several criteria, including clinical signs and change in body weight. Although the latter is used as an evaluation index in BSTs, there have been no reports on the internal changes that affect body weight during the test period. Therefore, we analyzed BST via pathological examination of the tested animals. Here, BSTs were performed for 176 batches using mice and 126 batches using of guinea pigs. Most of the gross findings could be classified into four lesion types (nodules, adhesions, ascites, no apparent signs), with only one vaccine inducing lesions that could not be classified into any of these four types. Histopathological examination revealed that the reactions caused by BST were pyogenic and/or granulomatous inflammation. Nodular or adhesive lesions comprised more severe pyogenic granulomatous inflammation than ascites or cases with no apparent gross lesions. These nodular or adhesive lesions were more frequently induced by vaccines that contained an adjuvant than by vaccines that did not contain an adjuvant. The cases with “exceptional” gross findings histologically presented severe necrosis of the hematopoietic system. Additional testing showed that these “exceptional” lesions were induced when a specific type of light liquid paraffin was injected along with other vaccine additives. Our results show that body weight loss and/or lesions during BST were induced by proinflammatory properties of the tested vaccines and that BST is a sensitive method for detecting unexpected effects of vaccine components.     

Frequency of enteropathogen detection in suckling and weaned pigs with diarrhea in Japan.

Fecal samples from suckling (n=153) and weaned (n=116) piglets with diarrhea in Japan were examined for shedding of viral, bacterial, and parasitic pathogens using culture, microscopic, and polymerase chain reaction methods. In suckling piglets, diarrhea was attributed to infection with a single etiologic agent in 60.8% of cases and with combinations of agents in 22.2%. In weaned piglets, diarrhea was attributed to a single etiologic agent in 43.1% and to combinations of agents in 47.4% of cases. Rotavirus was the most prevalent agent in suckling (67.3%) and weaned (65.5%) piglets. The detection of other pathogens was associated with age of the animals examined. Coccidia were predominantly isolated from suckling piglets, whereas Escherichia coli was found predominantly in weaned piglets. Although a relationship was not observed between detection rate of rotavirus and age of piglets, a single group of rotavirus was detected in 87.5% of suckling piglets whereas multiple groups were detected in 51.6% of weaned piglets. The results of this study confirm that diarrhea in piglets can, to a variable degree, be causally associated with multiple agents. Additionally, these results suggest reasons why this syndrome can be difficult to control.     

Performance of a foot-and-mouth disease virus reverse transcription-polymerase chain reaction with amplification controls between three real-time instruments.

The foot-and-mouth disease virus (FMDV) is a member of the picornavirus family, possessing an 8-kb single-stranded RNA genome of positive polarity. It is highly contagious among several livestock species and can lead to severe economic consequences, as evidenced by the UK outbreak in 2001. The usage of real-time polymerase chain reaction has facilitated rapid detection of FMDV. Several real-time PCR instruments are available with various capabilities, such as portability and high sample volume analysis. Primers and a dual-labeled TaqMan probe were optimized to detect a single, highly conserved 88-bp segment of the FMDV 3D (RNA polymerase) gene. To increase the confidence of the RT-PCR result, a positive amplification control was synthesized to detect potential false-positive results due to contamination if a wild-type virus is used as positive control. In addition, a preventative measure against false-negative results was developed in which endogenous beta actin mRNA is coamplified by RT-PCR. Assay performance was compared on the LightCycler1.2 (Roche), the SmartCyclerII (Cepheid), and the SDS 7900HT (ABI). These assays successfully identified the FMDV genome and beta actin mRNA from several sources of infected nasal and oral swabs, as well as probang samples.     

Fatigue of structural plywood under cyclic shear through thickness I: fatigue process and failure criterion based on strain energy

The fatigue behavior of plywood specimens under shear through thickness was examined on the basis of strain energy to obtain common empirical equations for the fatigue process and failure criterion under various loading conditions. Specimens were cut from commercial plywood panels of 9-mm thickness. Loading conditions were set as follows: a square waveform at a loading frequency of 0.5 Hz, a triangular waveform at 0.5 Hz, and a triangular waveform at 5.0 Hz. Peak stress applied was determined to be 0.5, 0.7, and 0.9 of static strength, that is, stress levels of 0.5, 0.7, and 0.9. The stress-strain relationships were measured throughout the fatigue test, and the strain energy was obtained at each loading cycle. Loading conditions apparently affected the relationship between stress level and fatigue life. On the other hand, the relationship between mean strain energy per cycle and fatigue life was found to be independent of loading conditions. Mean strain energy per cycle obtained as the fatigue limit was 5.85 kJ/m³ per cycle. Assuming that the accumulation of strain energy is a fatigue indicator, the fatigue process and failure criterion for the plywood specimens under the three loading conditions were commonly expressed by the relationship between cumulative strain energy and loading cycles.     

Pharmacokinetics of metoclopramide in calves with renal dysfunction

To clarify the effect of renal dysfunction on pharmacokinetics of the prokinetic agent metoclopramide (MCP), we administered intravenously 0.4 mg/kg MCP to healthy calves and calves subjected to right kidney vessel ligation (ligation) without or with a subsequent left nephrectomy (ligation plus removal). Plasma MCP concentration, glomerular filtration rate (GFR) and plasma prolactin level were measured by liquid chromatography-tandem mass spectrometry, simplified equation using iodixanol and enzyme-linked immunosorbent assay, respectively. Only in calves with ligation plus removal, plasma MCP concentrations were increased significantly 6, 8 and 12 hr after injection, showing that a negative correlation was observed between the plasma MCP concentrations and GFR value. A tendency to increase in plasma PRL concentration was noted also in these calves. In conclusions, plasma MCP concentrations depend on the GFR mode in calves, and its critical GFR value was estimated.     

Genetic variants of the unsaturated fatty acid receptor GPR120 relating to obesity in dogs

G protein-coupled receptor (GPR) 120 is an unsaturated fatty acid receptor, which is associated with various physiological functions. It is reported that the genetic variant of GPR120, p.Arg270His, is detected more in obese people, and this genetic variation functionally relates to obesity in humans. Obesity is a common nutritional disorder also in dogs, but the genetic factors have not ever been identified in dogs. In this study, we investigated the molecular structure of canine GPR120 and searched for candidate genetic variants which may relate to obesity in dogs. Canine GPR120 was highly homologous to those of other species, and seven transmembrane domains and two N-glycosylation sites were conserved. GPR120 mRNA was expressed in lung, jejunum, ileum, colon, hypothalamus, hippocampus, spinal cord, bone marrow, dermis and white adipose tissues in dogs, as those in mice and humans. Genetic variants of GPR120 were explored in client-owned 141 dogs, resulting in that 5 synonymous and 4 non-synonymous variants were found. The variant c.595C>A (p.Pro199Thr) was found in 40 dogs, and the gene frequency was significantly higher in dogs with higher body condition scores, i.e. 0.320 in BCS4–5 dogs, 0.175 in BCS3 dogs and 0.000 in BCS2 dogs. We conclude that c.595C>A (p.Pro199Thr) is a candidate variant relating to obesity, which may be helpful for nutritional management of dogs.