Considering the usefulness of the immunopotentiator from Pantoea agglomerans 1 (IP-PA1), which is a purified lipopolysaccharide (LPS) derived from symbiotic gram-negative bacteria of food crops, in controlling immunosuppression in poultry husbandry, in this study, we examined its immune-recovery effects in dexamethasone-treated stressed chicken models. Three-week-old chickens daily administered 10 μg/kg of dexamethasone for 35 days to induce stress showed more whole body weight loss; relative thymic, bursal, and splenic weight losses; and decrease in the number of peripheral blood lymphocytes, as compared with the control chickens on day 35; the IP-PA1-pretreated, dexamethasone-treated chickens showed reduced weight losses. Five- to eight-week-old chickens administered 5 mg/kg of dexamethasone showed excessive apoptosis of thymic and bursal lymphocytes 24 hr after a single dexamethasone treatment; apoptosis was inhibited in the IP-PA1-pretreated, dexamethasone-treated chickens. Chickens daily administered 10 μg/kg of dexamethasone for 35 days and injected with commercial Salmonella Enteritidis (SE) vaccine or sheep red blood cells (SRBC) on days 7 and 21 showed about 8- or 2-fold lower antibody production in response to SE or SRBC, respectively, as compared with the control chickens on day 35; the antibody production in response to SE or SRBC was increased in the IP-PA1-pretreated, dexamethasone-treated chickens. These results indicate that IP-PA1 exerts inhibitory effects on dexamethasone-induced immunosuppression and that it may be useful in controlling immunosuppression in poultry husbandry.
A 7-week-old SPF chicken inoculated at 4 weeks of age with chicken anemia virus was puffed up depressed and had ruffled feathers and a good body condition. Intestinal volvulus involving the jejunum and part of the duodenum forming two loops with one knob was observed. Microscopically, venous infarction of the obstructed loops, periportal and sublobular multifocal coagulative hepatic necrosis and granulomatous inflammation of the cecal tonsils were observed. Gram staining revealed no bacteria in hepatic tissue; however, gram-positive bacilli were detected in the necrotic debris in the intestinal lumen. Immunosuppression might have predisposed the chicken to intestinal and cecal tonsil infection that then progressed to volvulus. Loss of the mucosal barrier in infarction might allow bacterial toxins and vasoactive factors to escape into the systemic circulation (toxemia) and be responsible for the hepatic necrosis.
A pathogenic mutant of the Newcastle disease virus (NDV) was previously generated by passaging a non-pathogenic isolate from wild waterfowl. Velogenic mutant 9a5b (IVPI=2.67) contains three amino acid substitutions (128H, 495K and 573stop) in the hemagglutinin-neuraminidase (HN) protein, as compared with nonpathogenic waterfowl isolate 415/91 strain, and two of these (128H and 495K) were introduced after mesogenic 9a3b (IVPI=1.88). To investigate the role of the HN protein in NDV virulence, the function of HN protein such as neuraminidase (NA), Hemadsorption (HAd) and fusion promotion activities was examined by introducing the point mutations observed in passaged mutants into the HN gene cDNAs. In vitro functional assay using mutant protein expression demonstrated that the 128H substitution markedly increases NA activity and 573stop substitution increase NA and HAd activities. On the other hand, 495K substitution had little effect on any activities. These results indicate that a single amino acid substitution (128P to H) in the NDV HN protein affects the neuraminidase activity and is possibly correlated with the virulence.
Clinical courses of primary immune-mediated hemolytic anemia (pIMHA) in dogs are highly variable, however, limited information is available to predict their accurate prognoses. To evaluate the prognostic significance of clinical factors and to propose a scoring system to predict prognoses, the medical records of seventy-one dogs with pIMHA were reviewed. Overall mortality rate of dogs with pIMHA was 39% and most of the dogs died within 3 months from diagnosis. Sex, body weight, seasonality, packed corpuscular volume (PCV), platelet count (PLT), total plasma protein (TP), blood urea nitrogen, albumin, total bilirubin, sodium ion, prothrombin time, and fibrin/fibrinogen degradation products before immunosuppressive treatment can influence on survival time in dogs with pIMHA. A prognostic scoring system using a combination of sex, seasonality, PCV, PLT and TP can be statistically significant for raising the accuracy of prognostic prediction. Using the scoring system for prognostication in dogs with pIMHA may enable veterinarians to predict a prognosis easily and accurately.
We performed continuous renal replacement therapy (CRRT) on clinically healthy dogs to evaluate the effects of CRRT on hemodynamics. Heart rate, arterial blood pressure, and central venous pressure of the dogs (n=6) were recorded during the procedure, which was performed under general anesthesia. Throughout the CRRT, heart rate and arterial blood pressure were stable. Central venous pressure increased after CRRT termination but returned to the basal level within 30 min. In this study, hemodynamic alterations, including hypotension, hypertension, and arrhythmias, were not observed during CRRT. These observations suggest that the CRRT protocol used in the present study can be safely applied to clinical cases with acute renal failure.
The aim of this study was to determine whether socialized Jindo puppies would show different behavioral reactivity from non-socialized puppies. Puppies (n=12), 7 weeks of age, were divided into socialized and non-socialized groups. The socialized group from the 7th until 13th week after birth was provided a socialization program, and the non-socialized group was reared in a semi-isolated environment without being exposed to the program. At 13 weeks after birth, both groups were adopted by new families and raised as a family pet until adulthood. Both groups were tested in 5 behavioral tests at 7, 9, 11, 13 and 60 weeks of age, and their behavioral responses to the tests were recorded using video cameras. The contact, fearful and playful behaviors toward each behavioral test were scored on a scale of 1 to 5 points. Using all of the score data, a principal component analysis (PCA) extracted three primary factors: `social reactivity towards humans and a dog', `playful reactivity towards novel stimuli and a dog' and `fearful reactivity towards social stimuli'. The three extracted factors were compared between the socialized and non-socialized groups in each test session (weeks). Based on the results, the socialized Jindo puppies in the test session at the 9th week after birth, in contrast to the non-socialized puppies, exhibited a higher intensity of playful reactivity towards novel stimuli and a dog. However, there were no effects of the socialization program on the Jindo puppies in terms of social reactivity towards humans and a dog as well as fearful reactivity towards social stimuli.
We performed a thoracic X-ray examination of 56 Japanese macaques to obtain normal reference values for vertebral heart scale (VHS). Mean VHS was 10.25 ± 0.94v. In males, mean VHS was 10.56 ± 0.73v, with no significant correlation to age or weight. In contrast, mean VHS in females was 9.97 ± 1.03v, and tended to decrease with increasing age and weight. These findings will facilitate the diagnosis of cardiac disease in Japanese macaques in the future.
Feline chronic progressive polyarthritis is a rare immune-mediated disease that has only previously been reported in male cats. A one-year-old female cat was presented with anorexia, lassitude and lameness. The tarsal, carpal and elbow joints revealed swelling, pain, stiffness, crepitus and regional lymphadenopathy, and fever was present. The cat was clinically diagnosed with chronic progressive polyarthritis based on the fever, swelling of joints, imaging of erosive proliferative periosteal polyarthritis, positivity for antinuclear antibody, synovial fluid analyses and urinalyses. Both feline leukemia virus antigen and feline immunodeficiency virus antibody were positive. Using hair root DNA, polymerase chain reaction amplification targeting the sex-determining region on the Y chromosome gene amplified the fragment of DNA from a normal male cat, but not amplified from a normal female cat or the present cat. Accordingly, the present cat was classified as genetically female. Cyclosporine treatment was started, and the general condition and movement quickly improved and continued for 8 months post-diagnosis. This is the first report of chronic progressive polyarthritis in a female cat.
A 6-year-old, intact male Schnauzer was referred 2-days after accidental ingestion of baked garlic. Regenerative anemia (Hematocrit 22%) and the elevated methemoglobin (8.7%) concentration were detected upon hematological examination. Eccentrocytes, Heinz bodies and ruptured red blood cells were also noted on blood smear films, which were the results from the oxidative injury of the Allium species. The dog was hypertension (systolic mean 182 mmHg) concurrent with other clinical signs, such as vomiting and dark brown urination. Treatment with continuous oxygen, antioxidant drugs and antihypertensive therapy resulted in good progress. The dog was discharged 4 days after hospitalization. There were no remarkable findings in the follow up hematologic examination 24 days after discharge, but the dog still had a high blood pressure and continued on antihypertensive therapy. No recurrence was noted and the blood pressure returned to normal levels 4 months later.
Right lateral position (RL) chest radiograms were obtained from 27 heart disease-free rabbits, and cardiac sizes were compared according to body weight (<1.6 vs. ≥1.6 kg) and gender (female vs. male). Significant differences (P<0.05) were observed in RL-long axis (RL-LA) and RL-vertebral heart size (RL-VHS). RL-LA was 4.22 ± 0.25 and 4.48 ± 0.3, and RL-VHS was 7.55 ± 0.38 and 7.99 ± 0.58, in the <1.6 kg (n=12) and ≥1.6 kg (n=15) groups, respectively. These values should prove useful as new diagnostic indices for cardiac disease in rabbits.
Although Trypanosoma (Megatrypanum) theileri, a blood parasite of bovid species, is spread widely throughout the world, it has never been reported in Taiwan. When an anti-coagulated blood sample from febrile dairy cattle was directly smeared, no parasite was observed. However, a highly distinctive morphological feature of trypanosome appeared in baby hamster kidney (BHK) cell culture inoculated with non-thrown blood buffy coat. The different stages and typical ultrastructures of trypanosome were observed in our isolate. The isolate was subsequently identified as T. theileri by species-specific PCR assay (Tth625), 18S rDNA sequencing alignment and internal transcribed spacer of ribosomal genes (ITS) as a marker for molecular phylogenetic analysis. The first T. theileri isolate in Taiwan (TWTth1) could be periodically passaged in BHK cell culture for more than one year and retained good re-cryopreservation viability. The BHK culture method would be excellent for diagnostic isolation and maintenance long-term development of this parasite.
Current chemotherapeutic options for African trypanosomiasis in humans and livestock are very limited. In the present study, a total of 71 medicinal plant specimens from 60 plant species collected in Myanmar were screened for antitrypanosomal activity against trypomastigotes of Trypanosoma evansi and cytotoxicity against MRC-5 cells in vitro. The methanol extract of dried rootbark of Vitis repens showed the highest antitrypanosomal activity with IC50 value of 8.6 ± 1.5 μg/ml and the highest selectivity index of 24.4. The extracts of Brucea javanica, Vitex arborea, Eucalyptus globulus and Jatropha podagrica had also remarkable activity with IC50 values and selectivity indices in the range of 27.2-52.6 μg/ml and 11.4-15.1 respectively.
In the present study, we have characterized muscarinic receptor subtypes that mediate carbachol-induced Ca2+ sensitization of contraction in intestinal smooth muscle, using mutant mice lacking M2 or M3 muscarinic receptors or both receptor subtypes. In α-toxin-permeabilized muscle strips from wild-type (WT) mice, isometric tension responses to Ca2+ applied cumulatively (pCa 7.0-5.0) were increased when the muscarinic agonist carbachol (100 μM) was added to the medium, as judged from shifts of pCa-tension curves in both 50% effective concentration (EC50) and maximum response (Emax) of pCa-tension curve. In preparations from M2-knockout (KO) mice, pCa-tension curves were also shifted by carbachol (100 μM), and the extents of the EC50 and Emax changes resembled those observed in preparations from WT mice. In preparations from M3-KO or M2/M3-double KO mice, however, no significant changes in pCa-tension curves were obtained after carbachol application. The Gq/11-type G-protein inhibitor YM-254890 (1 μM) completely blocked the Ca2+ sensitization of contraction induced by carbachol in M2-KO or WT preparations. The results strongly support the idea that the muscarinic activation of Ca2+ sensitization in intestinal smooth muscles is mediated by the M3 muscarinic receptor coupled to Gq/11-type G-proteins, without any significant involvement of the other muscarinic receptor subtypes including M2.
Tick-borne encephalitis virus (TBEV) is a zoonotic agent that causes acute central nervous system (CNS) disease in humans. In this study, we examined the pathogenic process following intracerebral infection with the Oshima strain of TBEV in a mouse model. Intracerebral infection resulted in dose-dependent mortality, and all mice died following challenge with 102 PFU or more of the virus within 10 days. Acutely necrotic neurons and widespread inflammation were observed throughout the CNS. We therefore conclude that mortality following intracerebral infection results from a direct CNS pathology.
Many microorganisms produce extracellular polymers referred to collectively as "slime" or glycocalyx, and form biofilms on solid surfaces in natural ecosystems. Campylobacter jejuni, one of the most important foodborne pathogens, also has the ability to form biofilm on stainless steel, glass, or polyvinyl chloride in vitro. However, the issue of biofilm formation by Campylobacter species has not been extensively examined. The present study was performed to examine the mode of adhesion of C. jejuni to a smooth surface. When bacterial suspensions in Brucella broth were incubated in microplate wells with a glass coverslip, microcolonies 0.5~2 mm in diameter were formed on the coverslip within 2 hr from the start of incubation. These microcolonies gradually grew and formed a biofilm of net-like connections within 6 hr. Transmission electron microscopy indicated that massive amounts of extracellular material masked the cell surface, and this material bound ruthenium red, suggesting the presence of a polysaccharide moiety. Scanning electron microscopy indicated that the flagella acted as bridges, forming net-like connections between the organisms. To determine the genes associated with biofilm formation, aflagellate (flaA-) and flagellate but non-motile (motA-) mutants were constructed from strain 81-176 by natural transformation-mediated allelic exchange. The flaA- and motA- mutants did not form the biofilm exhibited by the wild-type strain. These findings suggest that flagella-mediated motility as well as flagella is required for biofilm formation in vitro.
The aim of the present study was to investigate the occurrence of Listeria monocytogenes in salami samples collected from production plants of the Marche Region, and to assess the end-product acceptability based on the former Italian regulations and European Commission (EC) Regulation N° 2073/2005. Based on the limits specified in the former Italian regulations, the percentage of non-acceptable samples was 34.3%, whereas based on the limits specified in EC Regulation N° 2073/2005, a lower percentage (17.1%) was seen. A similar trend was seen also when only the Ciauscolo salami were considered, with 45.2 and 27.4% of non-acceptable samples, respectively. No correlations were identified between occurrence of L. monocytogenes and the main parameters or the manufacturing processes.
Postoperative changes in endocrinological status and serum chemistry during the 4 years after transsphenoidal surgery (TSS) in 25 dogs with Cushing's disease were investigated in a prospective study. In all 25 dogs, Cushing's disease was diagnosed from resected pituitary tissues as a corticotroph adenoma in the anterior lobe of the pituitary. Prior to TSS, all 25 dogs showed hypercortisolemia. After TSS, the ACTH stimulation test showed continued low serum cortisol concentrations in 21 dogs (84%). In addition, the serum thyroid stimulating hormone concentrations decreased sequentially, while the serum T4 concentrations tended to increase due to the postoperative hormone substitution therapy utilized to avoid secondary hypothyroidism. In regard to serum chemistry, alkaline phosphatase (ALP), alanine aminotransferase (ALT) and total cholesterol are commonly increased in canine Cushing's disease. In this study, ALP, ALT and total cholesterol were increased in 23 cases (92%), 19 cases (76%) and 20 cases (80%), respectively. However, postoperatively, these concentrations gradually decreased. The postoperative serum concentrations of ALP at 1 year, that of ALT at six months to 2 years and that of total cholesterol over the course of the 4 years decreased significantly compared with the concentrations before TSS. These results show that TSS is an effective treatment for canine Cushing's disease and for long-term improvement of hypercortisolemia. Moreover, TSS is effective in improvement of hypercortisolism, such as increased concentrations of serum ALP, ALT and total cholesterol.
Two cases of bovine male pseudohermaphrodites (PH) were subjected for clinical investigation with transrectal ultrasonography, endocrinology with adoption of hCG-stimulation test, cytogenetics with analysis of sex chromosome and Y-specific DNA, and finally histological examination. Results were compared with normal calves. Case 1 was a 10-month-old calf with XX/XY chimeras, showing elevation of testosterone (T) levels, but no change in progesterone (P4) after hCG test, and possessed atrophied testes in the cavitas pelvis. Case 2 was an 18-month-old calf with SRY positive-XY chromosome, showing lower level of plasma T and P4 after hCG test, and possessed atrophied testes and undifferentiated genital ducts. Both cases possessed female-like external genitalia with similar pathological findings, however endocrinological and cytogenetical aspects were different each other.
Four commonly used pyrethroids (permethrin, bifenthrin, ethofenprox, and fenpropathrin) were orally administered to Sprague-Dawley rats for 5 days to study their effects on the liver cytochrome P450 (CYP) activities. Also Michaelis-Menten kinetics of the metabolic reactions catalyzed by liver CYPs were examined after adding these pyrethroids to the assay system to investigate their possible inhibitory effects on liver CYPs activities. These reactions included ethoxyresorufin O-deethylation, tolbutamide hydroxylation, bufuralol 1'-hydroxylation, and midazolam 4-hydroxylation, for CYP1A, 2C, 2D, and 3A activities, respectively. Results showed that oral administration of bifenthrin and ethofenprox highly induced CYP1A. The most potent inhibitors for CYP1A were fenpropathrin and cis-permethrin with Ki values of 3.71 & 3.87 μM, respectively. CYP2D was slightly inhibited by both of fenpropathrin and cis-permethrin (Ki values were 307.32 & 632.23 μM, respectively). On the other hand, none of CYP2C or 3A was inhibited by the tested pyrethroids. Since CYP1A may relate to biotransformation of many chemicals to reactive metabolites, bifenthrin and ethofenprox may potentiate mutagenicity of the chemicals through their inducing effects on CYP 1A. As permethrin and fenpropathrin were potent inhibitor for CYP1A, they may result in substantial accumulation of some chemicals. The resultant accumulation may lead to fatal toxicities in some case.
The developmental changes in the hepatic cytochrome P450 (CYP) content, mRNA expression of 12 hepatic CYP subtypes, and the enzyme activities of 5 hepatic CYP subfamilies in rats were investigated using non-treated male and female Sprague-Dawley rats of ages postnatal day (PD) 4, 16, 30 and 8 and 12 weeks. The hepatic proliferation kinetics was also determined by using the phospho-histone H3 (p-histon)-labeled hepatocyte index. The developmental changes in the enzyme activities of hepatic expression of CYP1A and CYP3A in rats were similar to those in humans, although there is no fetal-neonatal dominant CYP3A subtype in rat livers unlike human CYP3A7. On the other hand, the developmental pattern of expression of the CYP2C subfamily differed between humans and rats. Enzyme activity and mRNA expression of each hepatic CYP subtype in rats on PD 30 was similar to that after 8 weeks of age, except in the case of sex-dependent CYP subtypes. The p-histon-labeled hepatocyte index was approximately 10-fold higher in PD 30 rats than in 8-week-old rats. Therefore, the livers of juvenile rats, which have high hepatocellular proliferation activity and a sufficient amount of metabolic enzymes such as CYP, may be more sensitive to the cytotoxic and carcinogenic effects of chemicals than the livers of adult rats. Thus, our results on developmental difference of hepatic CYPs in juvenile rats are useful to identify underlying age-dependent susceptibility of chemical-induced toxicity, and to understand developmental change of chemical disposition.
On January 4, 2007, an emaciated mountain hawk-eagle was found in Kumamoto Prefecture, Japan. Highly pathogenic avian influenza (HPAI) virus subtype H5N1 was isolated from both tracheal and cloacal swabs of the dead bird. On January 13, an outbreak of HPAI, caused by H5N1 strain, occurred in a chicken farm in Miyazaki Prefecture. Within three weeks, three additional outbreaks had occurred (two in Miyazaki Prefecture and one in Okayama Prefecture). To investigate the relationship between the hawk-eagle isolate and chicken isolates, we studied the virus growth, pathogenicity, and phylogenetic information of this hawk-eagle isolate. The highest virus titer was found in the brain (107.25 EID50/g), followed by trachea and muscle (102.65 and 102.50 EID50/g, respectively). Sequence analysis at the hemagglutinin (HA) cleavage site of this isolate revealed a typical virulent-type sequence, R-R-R-K-K-R. Phylogenetic analysis demonstrated that the hawk-eagle isolate belongs to Qinghai Lake type virus group. A homology search of the HA gene also showed major similarity (more than 99%) to the Miyazaki and Okayama isolates in 2007 and also Korean isolates in 2006. These results suggest that Qinghai Lake type H5N1 HPAI virus was newly introduced from Asian Continent into Japan, and had already present in natural environment of Kyusyu district in the beginning of January 2007.
In February 2008, a feeder pig herd of the affected farm in Tochigi Prefecture, Japan, showed increasing respiratory symptoms; by April, the situation worsened with 12-16 pigs dying daily. Diagnostic tests revealed the presence of H1N2 subtype of swine influenza virus (SIV) and Pasteurella multocida from nasal swab and lung emulsion. Serological tests by hemagglutination inhibition method and enzyme-linked immunosorbent assay method (ELISA; imported from U.S.A.) indicated the spread of SIV into the pig herds of the affected farm around April 2008. The severe infection and subsequent damage were considered as a result of the combined infection of SIV (H1N2) and bacteria that may have been prevalent in the pig farm. Genetic homology search of sequences for the hemagglutinin (HA) and neuraminidase (NA) genes of A/swine/Tochigi/1/08 showed high homology to Japanese SIVs (H1N2) isolated in the 2000s. Therefore, we considered that Japanese SIV (H1N2) has established an independent stable lineage and participated in infecting pig populations as one of the factors of the pig respiratory disease complex. Consistent surveillance would contribute to clarifying the prevalence of dominant SIVs.
Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is an established gene amplification method for rapid diagnosis of various infectious diseases. In order to detect avian influenza viruses, particularly in field specimens, specific primers targeting the matrix gene were designed. Thirty-four virus samples, including isolates from wild and domestic avian hosts belonging to various geographical areas, were used to confirm the validity of the primers. All samples were confirmed to be positive in less than 1 hr. The RT-LAMP assay was also able to detect avian influenza virus in the various field samples, such as swabs, tissues, and feces. These results indicate that the developed RT-LAMP assay with uniquely designed primers is potentially useful in comprehensive avian influenza surveillance.