In this study, Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry was used to identify Mycobacterium bovis from cattle and buffalo tissue isolates from the North and South regions of Brazil, grown in solid medium and previously identified by Polymerase Chain Reaction (PCR) based on Region of Difference 4 (RD4), sequencing and spoligotyping. For this purpose, the protein extraction protocol and the mass spectra reference database were optimized for the identification of 80 clinical isolates of mycobacteria. As a result of this optimization, it was possible to identify and differentiate M. bovis from other members of the Mycobacterium tuberculosis complex with 100% specificity, 90.91% sensitivity and 91.25% reliability. MALDI-TOF MS methodology described herein provides successful identification of M. bovis within bovine/bubaline clinical samples, demonstrating its usefulness for bovine tuberculosis diagnosis in the future.
Staphylococcus aureus has the potential to opportunistically cause infectious diseases. The aim of this study was to determine the antimicrobial effects of novel graphene oxide (GO)-polyethylenimine (PEI)-based antisense yycG (ASyycG) on the inhibition of methicillin-resistant S. aureus (MRSA) biofilm formation. In current study, a novel GO-PEI-based recombinant ASyycG vector transformation strategy was developed to produce ASyycG. The mechanical features including zeta-potential and particle size distributions were evaluated by: GO; GO-PEI and GO-PEI-ASyycG. The recombinant ASyycG vector was transformed into MRSA cells, and the expression levels of the yycF/G and icaADB genes were determined and compared by quantitative real-time PCR (qPCR) assays. The recombinant ASyycG plasmids were subsequently modified with a gene encoding enhanced green fluorescent protein (ASyycG-eGFP) as a reporter gene, and the transformation efficiency was assessed by the fluorescence intensity. The biofilm biomass and bacterial viability of the MRSA strains were evaluated by crystal violet assay, colony-forming unit assays and confocal laser scanning microscopy. The results showed that the Z-average sizes of GO-PEI-ASyycG were much larger than those of GO or GO-PEI. The GO-PEI-based strategy significantly increased the efficiency of ASyycG transformation. The GO-PEI-ASyycG-transformed MRSA strain had the lowest expression levels of the biofilm formation-associated genes. Furthermore, GO-PEI-ASyycG suppressed biofilm aggregation and improved bactericidal effects on the MRSA after 24 hr of biofilm establishment. Our findings demonstrated that GO-PEI based antisense yycG RNA will be an effective method for management of MRSA infections.
Brown adipose tissue (BAT) contributes to non-shivering thermogenesis and plays an important role in body temperature control. The contribution of BAT thermogenesis to body temperature control in a non-cold environment was evaluated using developing hamsters. Immunostaining for uncoupling protein 1 (UCP1), a mitochondrial protein responsible for BAT thermogenesis, indicated that interscapular fat tissue had matured as BAT at day 14. When pups were placed on a thermal plate kept at 23°C, the body surface temperature decreased in day 7- and 10-day-old pups but was maintained at least for 15 min in 14-day-old pups, indicating that hamsters are unable to maintain their body temperature until around day 14 even in a non-cold environment. Body temperature maintenance was also evaluated in UCP1-deficient mice. BAT analysis showed that the UCP1 protein level in Ucp1+/− Hetero mice was 61.3 ± 1.4% of that in wild-type (WT) mice and was undetected in Ucp1−/− knockout (KO) mice. When 12-day-old pups were place on a thermal plate at 23°C, body surface temperature was maintained for at least 15 min in WT and Hetero mice but gradually dropped by 2.4 ± 0.2°C in 15 min in KO mice. It is concluded that BAT thermogenesis is indispensable for body temperature maintenance in pups of hamsters and mice, even in the non-cold circumstances. The early life poikilothermy and the later acquirement of homeothermy in hamsters may be because of the postnatal development of BAT.
LSm14A is a key innate immunity component of processing body (P-body) that mediates interferon-β (IFN-β) signaling by viral RNA. This is the first study to report chicken LSm14A (cLSm14A) cloning from blue eggshell layer, high tibia and frizzle chickens. The cLSm14A gene, encoding 461 amino acids, is highly homologous in the three types of chickens. The cLSm14A was extensively expressed in several tissues. The transcriptional level of cLSm14A was significantly increased in various stages of Newcastle disease virus (NDV) infection. In HEK293 cells, full length cLSm14A from blue eggshell layer was localized in the cytoplasm as dots. The results of this study indicated that cLSm14A is an important sensor that mediates innate immunity in chicken against NDV infections.
Porcine reproductive and respiratory syndrome (PRRS) is one of the major swine diseases responsible for a significant challenge in the global swine industry. The current PRRS inactivated vaccine only confers limited protection against PRRSV. Thus, using an appropriate adjuvant via a suitable administration route may help improve vaccine efficacy. In this study, the recombinant B subunit of the Escherichia coli heat-labile enterotoxin rLTB, was highly expressed in Pichia pastoris, through high-density fermentation. rLTB intranasal adjuvant properties were evaluated on an inactivated PRRS antigen in mice. Compared to the group immunized with solely PRRS antigen, a dose of 50 µg rLTB remarkably raised antigen-specific IgA antibodies at mucosal sites, and increased serum IgG antibodies, preferentially the IgG2a and IgG2b subclasses. Further, rLTB induced increases in Th1- (IFN-γ and IL-12) and Th17 (IL-6) cytokine profiles, but had little effect on Th2 cytokine profiles (IL-4 and IL-10). Moreover, there were no overt toxicities associated with intranasal rLTB administration. Our data provide evidence that the rLTB produced by P. pastoris fermentation portrays low toxicity, and its intranasal adjuvant effect involves immune system modulation to a Th1 profile.
Serum amyloid A (SAA) is an acute-phase protein indicative of inflammation. In murine colonic epithelial cells, lipopolysaccharide (LPS), a gram-negative bacterial antigen, strongly enhanced mRNA expression of SAA3, but not SAA1 or SAA2, suggesting that SAA3 might respond to bacterial infection in other epithelia. We examined SAA1/2 and SAA3 mRNA expression in murine alveolar epithelial cells exposed to LPS or the gram-positive bacterial antigen, lipoteichoic acid (LTA), using real-time PCR. LPS enhanced SAA3 mRNA expression at lower concentrations than did LTA, whereas SAA1/2 mRNA expression was not enhanced by either LPS or LTA. These results suggest that SAA3 expression is enhanced in lung epithelium upon bacterial infection as part of innate immunity, with higher sensitivity to LPS than to LTA.
Basic information related to the association between right heart echocardiographic parameters and invasive pulmonary artery pressure (PAP) in dogs with pulmonary hypetension (PH) is scarce. The aim of this study was to examine the association between conventional right heart echocardiographic parameters and invasive PAP by right heart catheterization (RHC) before and after PH. Five female beagle dogs regarded as clinically healthy were used. Echocardiography and RHC were conducted before and after creating chronic embolic pulmonary hypertension (CEPH) models. The acceleration time to ejection time ratio in pulmonary artery flow profile (AT/ET), the ratio of the pulmonary artery and aortic diameter in diastole (PA/Ao), the right pulmonary artery distensibility index by M-mode method (RPAD M-mode), the normalized right ventricular internal diameter in diastole (RVIDdn), and the normalized tricuspid annular plane systolic excursion (TAPSEn) were correlated with the invasive systolic PAP (sPAP), mean PAP (mPAP) and diastolic PAP (dPAP). Multiple linear regression analysis identified AT/ET and RVIDdn as independent predictors of sPAP, PA/Ao and RVIDdn as independent predictors of mPAP, and PA/Ao and RPAD M-mode as independent predictors of dPAP. AT/ET and PA/Ao had high sensitivity and specificity for predicting CEPH. In conclusion, AT/ET, PA/Ao, RPAD M-mode, RVIDdn and TAPSEn were significantly correlated with invasive PAP and alterations in PA/Ao or AT/ET might enable clinicians to predict PH, even if tricuspid regurgitation is not observed.
An 11-year-old, castrated male, Yorkshire Terrier was presented with acute vomiting after chicken bone ingestion. The dog had been diagnosed with hyperadrenocorticism previously and showed acute splenomegaly and signs of systemic inflammatory response syndrome during hospitalization. On diagnostic imaging, acute splenic vein thrombosis was found, concurrent with pancreatitis and gastritis. The spleen showed marked enlargement and hypoechoic lacy appearances on ultrasonography, mimicking splenic torsion. On the histopathologic report, only splenic hemorrhage and congestion with large splenic vein thrombosis were identified. After splenectomy, the dog completely recovered and was discharged.
Humans and dogs live very close together and share various pathogens causing zoonotic parasitoses like schistosomiasis. A previous population genetics study done for schistosomes in the Philippines suggested that there is a high transmission level of Schistosoma japonicum among humans and dogs proving that the latter are important reservoirs for this zoonotic parasite. A more sensitive and specific test detecting schistosome infection in dogs will therefore strengthen the zoonotic surveillance, which might help in the possible elimination of this ancient disease. In this study, recombinant thioredoxin peroxidase-1 (SjTPx-1) and tandem repeat proteins (Sj1TR, Sj2TR, Sj4TR, Sj7TR) previously tested on human and water buffalo samples were used to assess its diagnostic applicability to dogs. Fifty-nine dog serum and stool samples were collected in the schistosomiasis-endemic municipalities of Calatrava, Negros Occidental and Catarman, Northern Samar in the Philippines and examined using the ELISA as compared to microscopy and fecal sample-based PCR. Samples positive for Babesia gibsoni and Dirofilaria immitis were also used to check for cross-reaction. Results showed that SjTPx-1 (80% sensitivity, 92.3% specificity) and Sj7TR (73.3% sensitivity, 92.3% specificity) have good potentials for diagnosing S. japonicum infection in dogs. These diagnostic antigens will therefore improve the surveillance in the transmission of the parasites from dogs to humans.
A surveillance of Culicoides biting midges with light suction traps was conducted in the northern region of Honshu, main island of Japan, during the summers and autumns of 2009 and 2010. A total of 106 trap collections across 37 cattle farms were investigated for the structure and distribution of Culicoides species. Forty-thousand and one hundred forty-nine specimens of Culicoides biting midges were identified at the species level, and ≥19 species were included in the specimens. Culicoides oxystoma, which is a known major vector of Akabane virus (AKAV), appeared not to have expanded in northern Honshu during the surveillance. Of the potential AKAV vectors suggested by a previous laboratory experiment, C. tainanus and C. punctatus widely infested cowsheds across northern Honshu. The AKAV circulation was confirmed by serological surveillance of sentinel cattle in northern Honshu during the summer and autumn of 2010 and, consequently, >200 calves affected by the virus were identified as of spring 2011. Our surveillance demonstrated that C. tainanus and C. punctatus were widely spread and often dominated at cattle farms in/around the seroconverted regions, and our results thus suggest that these species played a critical role in the AKAV transmission in 2010. Because the distribution ranges of C. tainanus and C. punctatus cover almost all of mainland Japan, a potential risk of AKAV transmission might be expected even in areas outside the range of C. oxystoma.
In July 2017, twenty cattle of a free-grazing herd were found to be infested with leeches in the mouth. Main signs were bloody sialorrhea and/or a purple-red colour of the lower lip. Leeches, in a variable number (1 to 3) per animal, were found at the lingual frenulum or on the sublingual vestibular mucosa and were morphologically identified as Limnatis nilotica. To the best of our knowledge, this is the first report of cattle infestation by L. nilotica in Italy. Besides recalling the attention to leech infestation and suggesting its inclusion in the differential diagnosis of animals with suggestive signs, this short report also provides practitioners with easy-going morphological keys for proper diagnosis and discrimination among species.
A slaughtered 2-year-old female sika deer (Cervus nippon yesoensis) had diffusely distributed multinodular lesions on the serosal surface of the peritoneal cavity and several nodules in the pleural cavity. Histologically, they were composed of proliferating spindle-shaped neoplastic cells, arranged in a fascicular fashion. The cells in the invasive foci transitioned from a sarcomatoid to an epithelioid appearance. Immunohistochemically, both the spindle-shaped and epithelioid cells were at least focally positive for pancytokeratin, vimentin, calretinin, α-SMA, and desmin. From these findings, the deer was diagnosed with peritoneal sarcomatoid mesothelioma with metastasis to the pleural cavity. To our knowledge, this is the first reported case of peritoneal mesothelioma in a cervid species and the first case of mesothelioma in a sika deer.
Osteosarcoma (OSA) is the most common bone tumor in dogs. Protein phosphatase 2A (PP2A), an evolutionary conserved serine/threonine protein phosphatase, is a crucial tumor suppressor. SET is a PP2A inhibitory protein that directly interacts with PP2A and suppresses its phosphatase activity. SET has been reported as a contributor of wide range of human and dog tumor malignancies. However, the role of SET in canine OSA (cOSA) remains unknown. In this study, we investigated the role of SET in cOSA by using 2 cOSA cell lines: POS (primary origin) and HM-POS (metastatic origin). Knockdown (KD) of SET expression was noted to slightly suppress POS cell proliferation only. Furthermore, SET KD effectively suppressed colony formation ability of both POS and HM-POS cells. SET KD was observed to repress ERK1/2, mTOR, E2F1, and NF-κB signaling in HM-POS cells, whereas it inhibited only ERK1/2 signaling in POS. Further, it was observed that SET-targeting drug, FTY720, exerted anti-cancer effects in both POS and HM-POS cells. Moreover, the drug also enhanced the anti-cancer effect of cisplatin. The data suggested that a combination therapy, based on SET targeting drugs and cisplatin, could be a potent strategy for cOSA.
Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K) acts to inhibit protein translation through phosphorylating a specific substrate, eEF2. We previously found that the increased eEF2K expression in mesenteric artery mediates hypertension development in spontaneously hypertensive rats. More recently, we have revealed that a selective eEF2K inhibitor, A484954 induced vasorelaxation via opening inward rectifier K+ channel and activating β2-adrenergic receptor in smooth muscle of rat isolated mesenteric artery, which contributes to prevent noradrenaline-induced acute increase in blood pressure (BP). In this study, we further explored acute effects of A484954 on BP in rats, especially focusing the action on β-adrenergic receptor. We also examined whether A484954 affects contraction and heart rate (HR) of isolated heart. BP and HR were measured by a carotid cannulation method in rats. Isometric contraction and HR in rat isolated atria were also measured pharmacologically. A484954 potentiated adrenaline-induced decrease in diastolic BP (DBP) but not increase in systolic BP (SBP). A484954 potentiated isoproterenol-induced decrease in DBP but not SBP. Contrastingly, A484954 prevented a non-β-adrenergic receptor agonist, angiotensin II-induced increase in both SBP and DBP. In isolated left atria, A484954 caused contraction, which was prevented by a β-adrenergic receptor antagonist, propranolol. In isolated right atria, A484954 increased HR. In conclusion, we for the first time demonstrated that A484954 potentiates β-adrenergic receptor agonist-induced decrease in DBP possibly through vasorelaxation mediated via activating β2-adrenergic receptor. It was also demonstrated that A484954 causes contraction of rat isolated heart via activating β1-adrenergic receptor.
In healthy individuals, plasma glucose levels are maintained within a normal range. During fasting, endogenous glucose is released either through glycogenolysis or gluconeogenesis. Gluconeogenesis involves the formation of glucose-6-phosphate from a variety of precursors followed by its subsequent hydrolysis to glucose. Gluconeogenesis occurs in the liver and the kidney. In order to compare gluconeogenesis in canine liver and kidney, the activity and expression of the rate limiting enzymes that catalyze the fructose-6-phosphate and fructose 1,6-bisphosphate steps, namely, phosphofructokinase-1 (PFK-1) (glycolysis) and fructose bisphosphatase-1 (FBP-1) (gluconeogenesis), were examined. Healthy male and female beagle dogs aged 1–2 years were euthanized humanely, and samples of their liver and kidney were obtained for analysis. The levels of PFK-1 and FBP-1 in canine liver and kidney were assessed by enzymatic assays, Western blotting, and RT-qPCR. Enzyme assays showed that, in dogs, the kidney had higher specific activity of PFK-1 and FBP-1 than the liver. Western blotting and RT-qPCR data demonstrated that of the three different subunits (PFK-M, PFK-L, and PFK-P) the PFK-1 in canine liver mainly comprised PFK-L, whereas the PFK-1 in the canine kidney comprised all three subunits. As a result of these differences in the subunit composition of PFK-1, glucose metabolism might be regulated differently in the liver and kidney.
A 10-year-old male castrated Maltese was referred with clinical signs of hematuria, stranguria, and pollakiuria. The dog was diagnosed with sterile hemorrhagic cystitis with urethroliths and cystoliths. To remove the uroliths, the dog underwent retrograde urohydropropulsion followed by a cystotomy. The following day, persistent bleeding in the urinary bladder was identified with large hematoma, hematuria and anemia. In order to reduce bleeding, the dog received 10 mg/kg of tranexamic acid (TXA) intravenously. Immediately after TXA administration, the dog developed anaphylactic shock manifested by hypotension, hypothermia, tachycardia and a dull mentation. Thus, an emergency treatment including bolus injection of crystalloid, administration of dexamethasone and diphenhydramine, and oxygen supplementation was given, after which the dog quickly recovered within a few minutes.
An 11-year-old male Miniature Dachshund was referred for acute neurological deficits in the pelvic limbs. T2-weighted magnetic resonance imaging revealed that the spinal cord at the L1-2 intervertebral disc space was heterogeneously hyperintense in the sagittal plane and was mildly compressed from the ventral side by a small hypointense mass in the transverse plane. However, the lesion showed mass enhancement and severe spinal cord compression on post-contrast T1-weighted imaging. On three-dimensional myelography, a “golf tee sign” was observed around the mass. Therefore, we diagnosed an intradural extramedullary lesion. The mass was surgically removed and histologically diagnosed as a hemangiosarcoma. The “golf tee sign” observed on magnetic resonance myelography may be useful for distinguishing intradural extramedullary masses from intramedullary masses.
The purpose of this study was to clarify the effect of Bovine leukemia virus (BLV) infection on natural immunity in the bovine mammary gland and on the severity of clinical mastitis. We classified milk samples from clinical mastitic cows into BLV-positive (n=76) and BLV-negative (n=12). BLV-positive cows were further divided into cows with High BLV proviral load (H-PVL) (n=23) and Low BLV proviral load (L-PVL) (n=53). Severity of clinical mastitis was classified as MILD, MODERATE, or SEVERE. Multiple logistic regression analysis was performed on the host factors and environmental factors with severity of clinical mastitis as the objective variable. BLV proviral load (PVL) and season at onset of mastitis showed significant correlation with the severity of clinical mastitis. Binary logistic regression analysis was performed on natural immunity factors lactoferrin and lingual antimicrobial peptide (LAP) concentration in milk, with PVL as the objective variable. Of these natural immunity factors, LAP concentration in milk showed significant correlation with PVL. The results of the present study suggested that PVL and season are associated with severity of clinical mastitis, and that the immune function in the mammary gland is decreased in cows with H-PVL compared to that in cows with L-PVL.
Transmissible gastroenteritis (TGE), caused by transmissible gastroenteritis virus (TGEV), is a highly infectious disease in pigs. Vaccination is an effective approach to prevent TGEV infection. Here, we evaluated the potential of TGEV S1 as a DNA vaccine and porcine interleukin (pIL)-12 as an adjuvant in a mouse model. A DNA vaccine was constructed with the TGEV S1 gene to induce immune response in an experimental mouse model; pIL-12 was chosen as the immunological adjuvant within this DNA vaccine. The pVAX1-(TGEV-S1) and pVAX1-(pIL-12) vectors were transfected into BHK-21 cells and expressed in vitro. Experimental mice were separately immunized with each of the recombinant plasmids and controls through the intramuscular route. The lymphocytes isolated from the blood and spleen were analyzed for proliferation, cytotoxic activities, and populations of CD4+ and CD8+ cells. The titers of TGEV S1 in an enzyme-linked immunosorbent assay (ELISA) and TGEV neutralizing antibodies and the concentrations of interferon (IFN)-γ and IL-4 were also analyzed in the serum. The plasmids pVAX1-(TGEV-S1) and pVAX1-(pIL-12) could be expressed in BHK-21 cells, and the combination of pVAX1-(TGEV-S1) and pVAX1-(pIL-12) could induce a significant increase in all markers. pIL-12 could act as an immunological adjuvant in the DNA vaccine for TGEV-S1. Furthermore, the DNA vaccine prepared using TGEV-S1 and porcine IL-12 could induce excellent humoral and cellular immune responses.
Duck hepatitis A virus (DHAV) infection is characterized by severe hepatitis. In recent years, DHAV-A has become widespread in Asia and has led to economic losses. Conventional methods of DHAV-A detection must often be performed in the laboratory with inconvenience equipment. We have developed a rapid reverse transcription insulated isothermal (RT-iiPCR) technique for the on-site detection of DHAV-A based on the POCKITTM system in a convenient minitype device. We optimized the PCR primers and probes for the amplification of the DHAV-A 3C/3D genes, and successfully amplified a specific fragment of DHAV-A, but no fragment from 18 other duck pathogens. The limit of detection for viral RNA was 49 copies per reaction, and the sensitivity and specificity were each 100% in the analysis of 60 liver samples. By comparison, the sensitivities of RT-iiPCR was comparable in sensitivity to existing rRT-PCR. Furthermore, the RT-iiPCR results were 98.3% in agreement with those of the rRT-PCR, with a kappa value of 0.938. In conclusion, this new method not only offers a higher sensitivity and specificity than existing techniques, but also time-saving and better suited to field diagnoses because device is portable.
Akabane virus (AKAV) is an important insect-borne virus belonging to the genus Orthobunyavirus, the Peribunyaviridae family. An AKAV defined as GXDH 01 here, was isolated for the first time from blood from a sentinel goat in China in 2016, and its full-length open reading frames (ORFs) were sequenced in this study. Sequence analysis suggested that the isolate GXDH 01 probably had undergone a reassortment incident and acquired L segments from other strain originating from an attenuated vaccine, such as OBE-1. This study aims to provide more understanding as to the origin and epidemiology of AKAV in China.
Bovine viral diarrhea virus (BVDV) footprint has spread across the globe and is responsible for one of the most economically important diseases in cattle. In Japan, some regional surveillance and preventive measures to control bovine viral diarrhea (BVD) have been implemented. However, BVDV infection is poorly understood in cattle industries, and there is no systematic BVD surveillance system and control program. Kyushu is the center for raising beef cattle in Japan. Therefore, this study aimed to determine the BVDV infection using a slaughterhouse survey among beef cattle in Kyushu, Japan. A total of 1,075 blood samples were collected at two regional slaughterhouses in Miyazaki prefecture from December 2015 to June 2016. Antigen ELISA was used for detection of BVDV antigen in blood samples. Two samples showed positive results (2/1,075; 0.18%). BVDV RNA was extracted from positive blood samples; the sequence was determined and analyzed by the neighbor-joining method for construction of the phylogenetic tree. Phylogenetic analysis based on the 5’-UTR revealed that the two positive samples were grouped into the same subtype BVDV-1b in the BVDV-1 genotype, but the infected cattle belonged to two different farms. In conclusion, this is the first study to identify the presence of BVDV in a slaughterhouse survey in Kyushu. These findings suggest that a slaughterhouse survey is a useful tool for developing a surveillance system for monitoring infectious diseases in cattle.
The Feline coronavirus (FCoV) can lead to Feline infectious peritonitis (FIP), which the precise cause is still unknown. The theory of internal mutation suggests that a less virulent biotype of FCoV (FECV) would lead to another more pathogenic biotype (FIPV) capable of causing FIP. In this work, the 7b gene was amplified from 51 domestic cat plasma samples by semi-nested PCR and tested through phylogenetic and phylogeographical approaches. The 7b gene of Brazilian isolates displayed high conservation, a strong correlation between the geographic origin of the viral isolates and their genealogy, and its evolution was possibly shaped by a combination of high rates of nucleotide substitution and purifying selection.