A widespread deadly outbreak occurred in captive birds belonging to the family Turdidae in Italy. The present study was performed on 46 dead birds coming from 3 small decoy-bird breeders in central Italy. Only Turdus pilaris, Turdus iliacus, Turdus philomelos and Turdus merula were affected. No other species of bird held by these breeders died. A change of diet before the hunting season was reported from all breeders. Full necropsy of the animals and histological investigations of representative tissue samples were performed. Microscopical examination showed marked iron deposits in liver samples. Bacteriological investigations and molecular analysis to exclude bacterial and viral diseases were carried out. Contamination of food pellet samples by mycotoxins and analysis to detect heavy metal contaminants in food pellet samples were considered. An interesting result was the high iron content found in food pellets. It was higher than that considered suitable for birds, especially for species susceptible to development iron storage disease (ISD). Taken together, the results suggested an outbreak of ISD caused by the high iron content of food given to the birds before the hunting season. The high mortality recorded only in species belonging to the family Turdidae suggests a genetic predisposition in the affected birds.

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Scallop shell powder produced by calcination process − the average diameter of the powder particles being 20 µm (SSP) − was further ground into nano-sized particles, with average diameter of 500 nm, here designated CaO-Nano. Solution of CaO-Nano could inactivate avian influenza virus within 5 sec, whereas the solution of SSP could not even after 1 hr incubation. CaO-Nano solution could also inactivate Newcastle disease virus and goose parvovirus within 5 sec and 30 sec, respectively. The virus-inactivating capacity (neutralizing index: NI>3) of the solution was not reduced by the presence of 20% fetal bovine serum. CaO-Nano solution seems to be a good candidate of materials for enhancement of biosecurity in farms.

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A total of 21 (4.3%) enterohemorrhagic E. coli strains were isolated by biochemical tests and identification of the eae⁺stx1⁺stx2⁺ genotype from 490 stool samples obtained from calves with diarrhea during 1-year period from a major farm in Tehran, Iran. All of the strains showed resistance to ampicillin, ciprofloxacin, trimethoprim, streptomycin, chloramphenicol and tetracycline, while 19% showed resistance to gentamicin. Out of 21 EHEC strains, 11 (53%) harbored class 1 integron. Two different amplification products, which were approximately 750 and 1,700 bp in size, were obtained from amplified variable regions (in-F/in-R primers) in 3 (14.3%) and 4 (19%) of the EHEC isolates, which corresponded to dfrA7(dihydrofolate reductase type I) and dfrA1/aadA1(dihydrofolate reductase/aminoglycoside adenyltransferase) resistance gene cassettes, respectively, and this was confirmed by sequencing. Genotyping analysis revealed a total of 16 pulsotypes that corresponded to 16 isolates with the similarity indices of 62% and 30% for the most and least similar isolates, respectively, 9 of which harbored class 1 integron. Analysis of pulsotypes showed an extensive diversity among the isolates harboring integron, which is indicative of a lack of any significant genetic relatedness among the isolates. No obvious relation could be deduced between integron content and special pulsotypes. The little data available on the genotyping patterns of EHEC isolates from cattle and their resistance gene contents emphasize the need to establish genotyping databases in order to monitor and source track the source of emergence and spread of new resistant and integron-carrying genotypes.

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Escherichia coli resistance to quinolones has now become a serious issue in large-scale pig farms of China. It is necessary to study the dynamics of quinolone resistance in fecal Escherichia coli of pigs after antimicrobial administration. Here, we present the hypothesis that the emergence of resistance in pigs requires drug accumulation for 7 days or more. To test this hypothesis, 26 pigs (90 days old, about 30 kg) not fed any antimicrobial after weaning were selected and divided into 2 equal groups: the experimental (EP) group and control (CP) group. Pigs in the EP group were orally treated daily with 5 mg ciprofloxacin/kg of body weight for 30 days, and pigs in the CP group were fed a normal diet. Fresh feces were collected at 16 time points from day 0 to day 61. At each time point, ten E. coli clones were tested for susceptibility to quinolones and mutations of gyrA and parC. The results showed that the minimal inhibitory concentration (MIC) for ciprofloxacin increased 16-fold compared with the initial MIC (0.5 µg/ml) after ciprofloxacin administration for 3 days and decreased 256-fold compared with the initial MIC (0.5 µg/ml) after ciprofloxacin withdrawal for 26 days. GyrA (S83L, D87N/ D87Y) and parC (S80I) substitutions were observed in all quinolone-resistant E. coli (QREC) clones with an MIC ≥8 µg/ml. This study provides scientific theoretical guidance for the rational use of antimicrobials and the control of bacterial resistance.

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Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a dominant pathogenic bacterium in equine pneumonia. We developed a specific loop-mediated isothermal amplification (LAMP) method, which targets the gene encoding sorbitol-6-phosphate 2-dehydrogenase (sorD), for detecting S. zooepidemicus and examined the clinical efficacies of its use in combination with each of 3 DNA extraction methods easily used by veterinary practitioners, namely the Loopamp PURE DNA Extraction Kit, InstaGene Matrix and a conventional boiling method. The LAMP method plus the Loopamp PURE DNA Extraction Kit gave higher rates of positivity than the other combinations in both clinical and spiked samples containing clinically significant concentrations (>1 × 10⁴ CFU/ml) of S. zooepidemicus.

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We previously reported the presence of brown/beige adipocytes in the white fat depots of mature cattle. The present study examined the effects of dietary vitamin A on the expression of brown/beige adipocyte-related genes in the white fat depots of fattening cattle. No significant differences were observed in the expression of Ucp1 between vitamin A-deficient cattle and control cattle. However, the expression of the other brown/beige adipocyte-related genes was slightly higher in the mesenteric fat depots of vitamin A-deficient cattle. The present results suggest that a vitamin A deficiency does not markedly affect the expression of Ucp1 in white fat depots, but imply that it may stimulate the emergence of beige adipocytes in the mesenteric fat depots of fattening cattle.

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The corneal-protective effects of an artificial tear containing sodium hyaluronate (SH) and castor oil (CO) were evaluated on a porcine short-term dry eye model. Fresh porcine eyes with an intact cornea were treated with an artificial tear of saline, SH solution (0.1%, 0.5% or 1%), CO solution (0.5%, 1% or 5%) or a mixture solution containing 0.5% SH and 1% CO and then desiccated for 60, 90 or 180 min. To assess corneal damage, the eyes were stained with methylene blue (MB) or lissamine green (LG). The staining score of MB, absorbance of MB extracted from the cornea and staining density of LG increased significantly with increasing desiccation time in untreated and all artificial tear-treated eyes, although there were no significant differences in staining scores and absorbance of MB between eyes treated continuously with saline and 1% SH-treated ones at 60 and 90 min of desiccation or the mixture-treated eyes at 60 min of desiccation. No significant differences in the staining density of LG were also found between continuous saline-treated eyes and ones desiccated for 60 min and treated with 1% SH and the mixture. Mild cytoplasmic vacuolations were histopathologically observed in the basal and wing cells in eyes desiccated for 60 min and treated with 1% SH and the mixture. The mixture solution containing 0.5% SH and 1% CO has protective effects against corneal desiccation similar to those of 1% SH and would be helpful as an artificial tear.

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The efficacy of Malarone^® alone and in combination with doxycycline (DOXY) against Babesia gibsoni infections was examined in 8 dogs. In all dogs except one treated with Malarone^®, parasitemia decreased, and anemia improved soon after initiation of treatment. However, 3 of 4 dogs treated with Malarone^® relapsed, and relapse was inhibited in 2 of 4 dogs treated with Malarone^® and DOXY. All relapsed dogs responded well to the second treatment, but 1 dog relapsed again and did not respond to the third treatment. Malarone^® may be useful for acute stage of B. gibsoni infections, and at least second repeating treatment might be effective.

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The aim of the present study was to compare the impact on blood pressure and different metabolic parameters of a weight-loss program on obese dogs fed on a low-fat high-fibre diet and treated with and without mitratapide. The study sample consisted of 36 obese dogs, randomly assigned to a control group (n=17), which were fed on a low-fat high-fibre diet, and an intervention group (n=19), fed on the same diet and treated with mitratapide. Variables measured included body condition score, body weight, heart rate, systolic and diastolic blood pressures; total cholesterol, triglycerides and glucose levels; alanine aminotransferase and alkaline phosphatase activity, measured both at baseline (day 0) and at the end of the weight loss program (day 85). All the studied parameters had decreased in both groups at the end of the study; these being diastolic blood pressure, total cholesterol and alanine aminotransferase, significantly lower in dogs treated with mitratapide. The use of mitrapide in addition to low-fat high-fibre diet does not seem to offer any further useful effect in the loss of weight during the treatment of canine obesity. On the other hand, mitratapide seems to present certain beneficial effects on pathologies associated with obesity, these being mainly related to blood pressure, lipids and hepatic parameters.

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Toxoplasma gondii is a zoonotic protozoan parasite that infects humans and domestic animals. In this study, the seroprevalence of T. gondii antibodies was investigated using serum samples collected from 83 sheep, 146 goats and 37 cattle from a dozen subsistence farms in Bangladesh. Fifty-eight out of 83 sheep (69.9%), 89 out of 146 goats (61.0%) and 10 out of 37 cattle (27.0%) were seropositive for the parasite. Seroprevalence in young goats (<1 year old) was significantly lower than that of the adult goats (>1 year old). In contrast, seroprevalence for young and adult sheep was similar. These results indicate that acquired infection with T. gondii occurs in this region of Bangladesh, at least among goats.

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The presence of anti-erythrocyte autoantibodies in animals infected with various Babesia species is well reported. However, the pathogenesis of autoantibodies in babesiosis is poorly understood. Here, we demonstrated that anti-erythrocyte immunoglobulin (Ig) M and IgG were present in B. rodhaini-infected mice at 6 and 8 days after infection, respectively. Furthermore, we generated monoclonal antibodies against erythrocyte antigen from B. rodhaini-infected mice. Five clones were generated. By Western blotting analysis using whole erythrocyte antigens, one clone reacted with a broad band around 90–150 kDa, and the 2 clones reacted with a band larger than 150 kDa. B. rodhaini-infected mice and/or autoreactive monoclonal antibodies established in this study might be a powerful tool for in vivo pathogenesis studies of autoantibody development in infectious diseases.

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A nationwide survey of Babesia gibsoni using Haemaphysalis longicornis collected from dogs and cats in Japan was conducted using molecular methods. A total of 1,341 H. longicornis, including 305 females, 14 males, 332 nymphs and 690 larvae (153 pools) from 44 prefectures, were examined by B. gibsoni-targeted PCR. Partial sequence analysis revealed that 12 of 13 positive samples sequenced, including samples from Tottori, Hiroshima, Yamaguchi, Tokushima, Ehime and Oita prefectures (all in western Japan), were identical to B. gibsoni, and 1 sample from Kyoto Prefecture was most closely related to a Babesia species recently detected from feral raccoons in Hokkaido. H. longicornis is a candidate for transmission vector tick of the new Babesia species.

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A layer of epithelial cells prevents the invasion of bacteria and the entry of foreign substances into the underlying tissue. The disruption of epithelial tight junctions initiates and exacerbates inflammation. However, the precise mechanism underlying the disruption of the epithelial tight junction remains unclear. The activation of protease-activated receptor 2 (PAR2) by serine proteases produced by some bacteria and mast cells contributes to inflammation in many tissues. In the present study, we tested the hypothesis that PAR2 activation affects the structure and function of tight junctions in Madin-Darby canine kidney (MDCK) cells. Although the application of a PAR2-activating peptide, PAR2-AP, from the apical side of MDCK cells failed to modify the transepithelial resistance (TER), its application from the basal side markedly suppressed the TER. In 3-dimensional cultures of MDCK cells expressing the mCherry-tagged PAR2, a lateral localization of PAR2 was observed. The application of PAR2-AP from the basal side changed the localization of the tight junctional protein, zonula occludin-1. Furthermore, PAR2-AP induced the phosphorylation of p38 MAP kinase. A p38 MAP kinase inhibitor, SB202190, inhibited PAR2-AP-induced changes in TER. Our results suggest that the activation of PAR2 leads to the disruption of tight junctions and increases the barrier permeability through the activation of p38 MAPK, which may cause the initiation and exacerbation of inflammation.

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SET is an endogenous protein phosphatase 2A (PP2A) inhibitor and is associated with a poor prognosis in human leukemia. Previously, we reported increased SET protein levels in canine lymphoma cell lines and the potential therapeutic application of SET antagonists in canine lymphoma. Here, we found that canine cells express several isoforms of the SET protein. We cloned 4 isoforms of SET, named SETα, β, γ and δ. Genomic BLAST showed that the SET genes are located on chromosomes X, 7, 1 and 8, respectively. An immunofluorescent study showed nuclear localization of SETα and β, and nuclear and cytosolic localization of SETγ and δ. We confirmed that SETα and β possess the ability to associate with PP2A. Our data reveal the existence of unique SET isoforms that should be taken into account in SET-targeting drug development studies in dogs.

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We report group B Betacoronavirus infection in little Japanese horseshoe bats in Iwate prefecture. We then used reverse-transcription PCR to look for the coronavirus RNA-dependent RNA polymerase gene in fecal samples collected from 27 little Japanese horseshoe bats and found eight were provisionally positive. We had a success in the nucleotide sequencing of six of the eight positive samples and compared them with those of authentic coronaviruses. We found that these six samples were positive in coronavirus infection, and they belonged to the group B Betacornavirus by phylogenetic analysis. Virus isolation using the Vero cell culture was unsuccessful. Pathogenic trait of these bat coronaviruses remained unexplored.

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Environmental stress affects various parts of mammals typically through the circulation of stress hormones. It has been identified as one of the possible reasons for male reproductive difficulties, but the complex mechanisms responsible for stress-induced reproductive suppression are poorly understood. Here, we examined the relationship between chronic environmental stress and hypothalamic kisspeptin, a recently discovered upstream regulator of the reproductive endocrine feedback system. We studied male mice under an unpredictable chronic stress procedure to replicate the situation of animals under chronic stress. Histological and immunohistochemical analyses were performed focusing on kisspeptin neurons in the arcuate hypothalamic nucleus (ARC) and DNA fragmented cells in seminiferous tubules. Although the ARC was not morphologically altered in either the stressed or non-stressed group, granular kisspeptin immunoreactivities decreased slightly in the stress group. In the testes of the stress group, several signs of testicular degeneration were observed, including increased numbers of ssDNA-positive cells per seminiferous tubule, thinning, vacuoled seminiferous epithelia and multinucleated giant cells. The decreases in kisspeptin in the stress group might be due to other hypothalamic peptides, such as corticotropin-releasing hormone and leptin, whose receptors are known to coexpress in the ARC. In addition, environmental stress directly and indirectly affects testicular function through stress hormones and gonadotropins. In summary, our findings enhance the understanding of stress-induced reproductive suppression possibly mediated by kisspeptin in the ARC.

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This study was to investigate the localization and distribution of eosinophils (EOS) in the bovine oviduct throughout the estrous cycle. Histological studies revealed more abundant EOS in the infundibula of the oviducts ipsilateral to the preovulatory dominant follicle and the ovulated ovary. The number of EOS was higher in the infundibula of the oviducts ipsilateral to the ovulated ovary than those of the oviducts contralateral to the ovulated ovary. The infundibula of the oviducts ipsilateral to the preovulatory dominant follicle had higher number of EOS than those of the oviducts ipsilateral to the mid-cycle corpus luteum. The number of EOS in the isthmus, but not in the ampulla, was higher in the outer layers (tunica muscularis and tunica serosa) than in the inner layers (tunica mucosa and tunica submucosa) during the estrous cycle. Thus, the EOS number varied with the region of the bovine oviduct, with greater number in the infundibula of the oviduct ipsilateral to the ovulated ovary, suggesting the impact of ovulation.

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Interleukin 18 (IL-18) plays an important role in the T-helper-cell type 1 immune response against intracellular parasites, bacteria and viral infections. It has been widely used as an adjuvant for vaccines and as an anticancer agent. However, IL-18 protein lacks a typical signal sequence and requires cleavage into its mature active form by caspase 1. In this study, we constructed mammalian expression vectors carrying cDNA encoding mature canine IL-18 (cIL-18) or mouse IL-18 (mIL-18) fused to the human IL-2 (hIL-2) signal sequence. The expressed proIL-18 proteins were processed to their mature forms in the cells. The supernatants of cells transfected with these plasmids induced high interferon-γ production in canine peripheral blood mononuclear cells or mouse splenocytes, respectively, indicating the secretion of bioactive IL-18. Using reverse genetics, we also generated a recombinant canine distemper virus that expresses cIL-18 or mIL-18 fused to the hIL-2 signal sequence. As expected, both recombinant viruses produced mature IL-18 in the infected cells, which secreted bioactive IL-18. These results indicate that the signal sequence from hIL-2 is suitable for the secretion of mature IL-18. These recombinant viruses can also potentially be used as immunoadjuvants and agents for anticancer therapies in vivo.

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We developed a concise and broadly applicable method for accurate genomic sequencing of North American genotype (NA-type) porcine reproductive and respiratory syndrome viruses (PRRSVs) that overcomes high genetic variability of the viruses. The method, designated “combination of consensus oligonucleotide reverse transcription and multiple displacement amplification” (CORT-MDA), involves reverse-transcription of viral RNA followed by shotgun sequencing after amplification using only 11 degenerate oligonucleotide primers; these primers were designed against consensus regions within the open reading frames of the 124 NA-type PRRSV strains with reported full-length genomic sequences. Sequencing of the 192 shotgun clones generated per virus showed 80% to 94% coverage on the reported PRRSV genomic sequence, such that only 2 or 3 unread regions had to be resequenced after PCR amplification using custom primers. Direct sequencing of RT-PCR products confirmed absolute consistency between sequences determined by the CORT-MDA method and those from RT-PCR. These results suggest that our method is applicable to diverse NA-type viruses.

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Since late 2013, several outbreaks of porcine epidemic diarrhea virus (PEDV) infection have emerged in Taiwan. Suckling piglets under 2 weeks of age showed severe vomiting and watery yellowish diarrhea with morbidity and mortality ranging from 80 to 100% and 90 to 100%, respectively. A total of 68 samples from 25 pig farms were confirmed as positive for PEDV and negative for rotavirus and transmissible gastroenteritis virus by reverse transcription PCR, and the partial S gene of PEDV was analyzed. Phylogenetic analysis places all 18 Taiwanese PEDV isolates collected during this outbreak in the same clade as the US strains of PEDV. This novel PEDV is prevailing and currently causing severe outbreaks in Taiwan.

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A strain of equine herpesvirus type 1 (EHV-1) was isolated from zebra. This strain, called “zebra-borne EHV-1”, was also isolated from an onager and a gazelle in zoological gardens in U.S.A. The full genome sequences of the 3 strains were determined. They shared 99% identities with each other, while they shared 98% and 95% identities with the horse derived EHV-1 and equine herpesvirus type 9, respectively. Sequence data indicated that the EHV-1 isolated from a polar bear in Germany is one of the zebra-borne EHV-1 and not a recombinant virus. These results indicated that zebra-borne EHV-1 is a subtype of EHV-1.

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In the winter of 2010–2011, an outbreak of highly pathogenic avian influenza virus (HPAIV) infection occurred in wild and domestic birds in Japan. Tufted ducks were found dead in an urban area of Toyota City, Koriyama, Fukushima Prefecture. Two tufted ducks were examined histopathologically, immunohistochemically and molecularly. Gross findings included marked dark-red clotted blood in the pectoral muscles and multifocal hemorrhages on the serous membranes. Microscopically, non-suppurative meningoencephalitis, multifocal to coalescing pancreatic necrosis and severe pulmonary congestion were observed. HPAIV antigen was detected in the malacic areas, neuronal, glial and ependymal cells, pulmonary capillary endothelial cells and epithelium of pulmonary bronchioles, necrotic pancreatic acini and degenerated cardiac myocytes. The HPAIV isolate was genetically classified into clade 2.3.2.1 group A. The broad distribution of virus antigen in brain and pulmonary tissues associated with HPAIV spontaneous infection in tufted ducks might be useful in understanding its pathogenesis in nature.

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A 4-year and 2-month-old male capybara (Hydrochoerus hydrochaeris) was diagnosed with squamous cell carcinoma on the buttocks after chronic recurrent dermatosis. The capybara was euthanized, examined by computed tomography and necropsied; the tumor was examined histologically. Computed tomography showed a dense soft tissue mass with indistinct borders at the buttocks. Histological examination of the tumor revealed islands of invasive squamous epithelial tumor cells with a severe desmoplastic reaction. Based on the pathological findings, the mass was diagnosed as a squamous cell carcinoma. This is the first study to report squamous cell carcinoma in a capybara.

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