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26.  Decreased plasma Chromogranin A361-372 (Catestatin) but not Chromogranin A17-38 (Vasostatin) in female dogs with bacterial uterine infection (pyometra) 
Pyometra often induces systemic inflammatory response syndrome (SIRS) and early diagnosis is crucial for survival. Chromogranin A (CgA) is a neuroendocrine secretory protein that is co-released with catecholamines from the adrenal medulla and sympathetic nerve endings. A prognostic value of CgA has been found in humans that are critically ill or that have SIRS associated with infection. CgA has not yet been studied in dogs with bacterial infection. The aim of the study was to investigate CgA, measured by Chromogranin A361-372 (Catestatin; Cst) and Chromogranin A17-38 (Vasostatin; VS) in healthy dogs and in dogs with pyometra.
Fifty dogs with pyometra, sampled prior to surgery and 64 healthy female dogs were included. In 19 pyometra cases, blood samples were also collected postoperatively. Concentrations of Cst and VS were measured in heparinised plasma and Cst also measured in EDTA plasma, by in-house radioimmunoassays. Student’s t-test and Wilcoxon two-sample test was used to test for differences between dog groups. Pre- and postoperative samples in dogs with pyometra were analysed by paired t-test. Pearson correlation was used to investigate associations of laboratory variables and hospitalization. P < 0.05 was considered significant.
Concentrations of Cst were decreased in pyometra dogs (mean ± SE, 1.01 ± 0.05 nmol/L) compared to healthy dogs (mean ± SE, 1.70 ± 0.03 nmol/L) (p ≤ 0.0001). VS concentrations did not differ significantly between dogs with pyometra (0.40 ± 0.04 nmol/L) and healthy dogs (0.42 ± 0.03 nmol/L). Mean ± SE pre- and postoperative concentration of Cst (1.0 ± 0.04 nmol/L and 0.9 ± 0.2 nmol/L) and VS (0.36 ± 0.04 nmol/L and 0.36 ± 0.04 nmol/L) in dogs with pyometra did not differ significantly. Neither Cst nor VS concentrations were associated with duration of hospitalization and were not significantly different in the four dogs with pyometra that had prolonged (≥3 d) postoperative hospitalization.
Concentrations of Cst, but not VS, were decreased in pyometra. Cst and VS concentrations before and after ovariohysterectomy did not differ significantly and were not associated with duration of hospitalization. Further studies are warranted to evaluate a possible diagnostic or prognostic value for Cst and VS.
Electronic supplementary material
The online version of this article (doi:10.1186/s12917-015-0328-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4318355  PMID: 25636335
Pyometra; Chromogranin A; Catestatin; Vasostatin; C-reactive protein; Biomarker; Dog; Uterine infection
27.  Skin malformations in a neonatal foal tested homozygous positive for Warmblood Fragile Foal Syndrome 
Skin malformations that resembled manifestations of Ehlers-Danlos-Syndrome were described in a variety of domestic animals during the last century as cutis hyperelastica, hyperelastosis cutis, dermatosparaxis, dermal/collagen dysplasia, dermal/cutaneous asthenia or Ehlers-Danlos-like syndrome/s. In 2007, the mutation responsible for Hereditary Equine Regional Dermal Asthenia (HERDA) in Quarter Horses was discovered. Several case reports are available for similar malformations in other breeds than Quarter Horses (Draught Horses, Arabians, and Thoroughbreds) including four case reports for Warmblood horses. Since 2013, a genetic test for the Warmblood Fragile Foal Syndrome Type 1 (WFFS), interrogating the causative point mutation in the equine procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1, or lysyl hydroxylase 1) gene, has become available. Only limited data are available on the occurrence rate and clinical characteristics of this newly detected genetic disease in horses. In humans mutations in this gene are associated with Ehlers-Danlos Syndrome Type VI (kyphoscoliotic form).
Case presentation
This is the first report describing the clinical and histopathological findings in a foal confirmed to be homozygous positive for WFFS. The Warmblood filly was born with very thin, friable skin, skin lesions on the legs and the head, and an open abdomen. These abnormalities required euthanasia just after delivery. Histologic examination revealed abnormally thin dermis, markedly reduced amounts of dermal collagen bundles, with loosely orientation and abnormally large spaces between deep dermal fibers.
WFFS is a novel genetic disease in horses and should be considered in cases of abortion, stillbirth, skin lesions and malformations of the skin in neonatal foals. Genetic testing of suspicious cases will contribute to evaluate the frequency of occurrence of clinical WFFS cases and its relevance for the horse population.
PMCID: PMC4327794  PMID: 25637337
PLOD1; Equine procollagen-lysine; 2-oxoglutarate 5-dioxygenase 1; LH1; Lysyl hydroxylase 1; Ehlers-Danlos syndrome; Dermatosparaxis; Dermal hyperfragility; Horse; Equine; Connective tissue; Hereditary
28.  Seroprevalence and molecular characteristics of hepatitis E virus in household-raised pig population in the Philippines 
Hepatitis E virus (HEV) infection is a significant public health concern in Asia, and swine is an important source of sporadic HEV infection in human. However, no epidemiological data are available regarding HEV infection among the swine or human population in the Philippines. To assess the HEV infection status among pigs in rural areas, we investigated the molecular characteristics and seroprevalence of HEV among household-raised pigs in San Jose, Tarlac Province, the Philippines.
Serum and rectal swab samples were collected from 299 pigs aged 2–24 months from 155 households in four barangays (villages) between July 2010 and June 2011. Enzyme-linked immunosorbent assay (ELISA) revealed that 50.3% [95% confidence interval (CI) 44.5–56.2%] and 22.9% (95% CI 18.2–28.1%) of pigs tested positive for anti-HEV IgG and IgM, respectively. HEV RNA was detected in the feces of 22 pigs (7.4%, 95% CI 4.7–10.9%). A total of 103 households (66.5%, 95% CI 58.4–73.8%) had at least one pig that tested positive for anti-HEV IgG or IgM or HEV RNA. The prevalence of anti-HEV IgG and IgM in breeding pig (8–24 months) were higher than that in growing pigs (2–4 months) (p < 0.0001 and p = 0.008, respectively). HEV RNA was more frequently detected in 2–4-month-old pigs (9.2%, 95% CI 5.4–14.6%) than in ≥5-month-old pigs (4.8%, 95% CI 1.1–8.5%) without statistical significance (p = 0.142). HEV RNA showed 0–27.6% nucleotide difference at the partial ORF2 gene among the detected viruses, and a majority of them belonged to subtype 3a (20/22, 90.9%).
We found a high prevalence of HEV antibodies in the household-raised pig population in rural areas of the Philippines, which indicates the potential risk of HEV infection among local residents. Only genotype 3 of HEV was observed, and genetically diverse strains of HEV were found to be circulating in pigs in this study.
Electronic supplementary material
The online version of this article (doi:10.1186/s12917-015-0322-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4312430  PMID: 25622684
Hepatitis E virus; Household-raised pig; Seroprevalence; Genotype 3; Philippines
29.  Development of learning objectives for neurology in a veterinary curriculum: Part II: Postgraduates 
Specialization in veterinary medicine in Europe is organized through the Colleges of the European Board of Veterinary Specialization. To inform updating of the curriculum for residents of the European College of Veterinary Neurology (ECVN) job analysis was used. Defining job competencies of diploma holders in veterinary neurology can be used as references for curriculum design of resident training. With the support of the diplomates of the ECVN and the members of the European Society of Veterinary Neurology (ESVN) a mixed-method research, including a qualitative search of objectives and quantitative ranking with 149 Likert scale questions and 48 free text questions in 9 categories in a survey was conducted. In addition, opinions of different groups were subjected to statistical analysis and the result compared.
A return rate of 62% (n = 213/341) was achieved. Of the competencies identified by the Delphi process, 75% objectives were expected to attain expert level; 24% attain advanced level; 1% entry level. In addition, the exercise described the 11 highly ranked competencies, the 3 most frequently seen diseases of the central and peripheral nervous systems and the most frequently used immunosuppressive, antiepileptic and chemotherapeutic drugs.
The outcomes of this “Delphi job analysis” provide a powerful tool to align the curriculum for ECVN resident training and can be adapted to the required job competencies, based on expectations. The expectation is that for majority of these competencies diplomates should attain an expert level. Besides knowledge and clinical skills, residents and diplomates are expected to demonstrate high standards in teaching and communication. The results of this study will help to create a European curriculum for postgraduate education in veterinary neurology.
Electronic supplementary material
The online version of this article (doi:10.1186/s12917-014-0314-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4323235  PMID: 25622644
Veterinary education; Curriculum; Learning objectives; Neurology; Postgraduate; Diplomate; Resident; ECVN; ESVN; Europe
30.  Population structure and antimicrobial susceptibility of Pseudomonas aeruginosa from animal infections in France 
Pseudomonas aeruginosa is a major human pathogen, which also affects animals. It is thought that P. aeruginosa has a non-clonal epidemic population structure, with distinct isolates found in humans, animals or the environment. However, very little is known about the structure of the P. aeruginosa population from diseased animals. Data on antimicrobial resistance are also scarce.
Thirty-four already registered and 19 new MLST profiles were identified. Interestingly, a few clones were more prevalent, and clones associated to human outbreaks were also detected. Multidrug resistance phenotypes were overall rare.
We highlight the non clonal structure of the population and show a higher prevalence of specific clones, possibly correlating with higher pathogenicity. The low proportion of antimicrobial resistance contrasts with the high resistance rate of human isolates.
Electronic supplementary material
The online version of this article (doi:10.1186/s12917-015-0324-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4307146  PMID: 25604773
Pseudomonas aeruginosa; Animal; MLST; Antimicrobial susceptibility
31.  Seroprevalence and risk factors of Chlamydia abortus infection in free-ranging white yaks in China 
Chlamydia is gram-negative obligate bacteria which causes a wide variety of diseases in humans and animals. To date, there are a few reports about the seroprevalence of Chlamydia and the risk factors associated with Chlamydia infection in yaks in the world. In this study, 974 blood samples were collected from white yaks (Bos grunniens) in Tianzhu Tibetan Autonomous County, Gansu province, northwest China from June 2013 to April 2014.
Antibodies against Chlamydia abortus were examined by the indirect hemagglutination (IHA) test, and 158 of 974 (16.22%) white yaks were seropositive for C. abortus antibodies at the cut-off of 1:16. The risk factors associated with seroprevalence were evaluated by a multivariate logistic regression analysis. Region, gender and age of white yak were left out of the final model, due to its insignificance in the logistic regression analysis (P > 0.05). However, season was considered as a major risk factor associated with C. abortus infection in white yaks.
To our knowledge, this is the first survey of C. abortus seroprevalence in white yaks in China, which extends the host range for C. abortus and has important implications for public health and the local Tibetan economy.
PMCID: PMC4308933  PMID: 25601354
Chlamydia abortus; White yaks; Seroprevalence; Tibetans; China
32.  Stability of 3-bromotyrosine in serum and serum 3-bromotyrosine concentrations in dogs with gastrointestinal diseases 
3-Bromotyrosine (3-BrY) is a stable product of eosinophil peroxidase and may serve as a marker of eosinophil activation. A gas chromatography/mass spectrometry method to measure 3-BrY concentrations in serum from dogs has recently been established and analytically validated. The aims of this study were to determine the stability of 3-BrY in serum, to determine the association between peripheral eosinophil counts and the presence of an eosinophilic infiltrate in the gastrointestinal tract, and to compare serum 3-BrY concentrations in healthy dogs (n = 52) and dogs with eosinophilic gastroenteritis (EGE; n = 27), lymphocytic-plasmacytic enteritis (LPE; n = 25), exocrine pancreatic insufficiency (EPI; n = 26), or pancreatitis (n = 27).
Serum 3-BrY concentrations were stable for up to 8, 30, and 180 days at 4°C, −20°C, and −80°C, respectively. There was no significant association between peripheral eosinophil count and the presence of eosinophils in the GI tissues (P = 0.1733). Serum 3-BrY concentrations were significantly higher in dogs with EGE (median [range] = 5.04 [≤0.63-26.26] μmol/L), LPE (median [range] = 3.60 [≤0.63-15.67] μmol/L), and pancreatitis (median [range] = 1.49 [≤0.63-4.46] μmol/L) than in healthy control dogs (median [range] = ≤0.63 [≤0.63-1.79] μmol/L; P < 0.0001), whereas concentrations in dogs with EPI (median [range] = 0.73 [≤0.63-4.59] μmol/L) were not different compared to healthy control dogs.
The present study revealed that 3-BrY concentrations were stable in serum when refrigerated and frozen. No relationship between peripheral eosinophil count and the presence of eosinophils infiltration in the GI tissues was found in this study. In addition, serum 3-BrY concentrations were increased in dogs with EGE, but also in dogs with LPE and pancreatitis. Further studies are needed to determine whether measurement of 3-BrY concentrations in serum may be useful to assess patients with suspected or confirmed EGE or LPE.
PMCID: PMC4299803  PMID: 25595676
3-bromotyrosine; Canine; Eosinophilic gastroenteritis; Stability
33.  Prevalence of Toxoplasma gondii antibodies in domestic donkeys (Equus asinus) in Durango, Mexico slaughtered for human consumption 
Nothing is known about Toxoplasma gondii prevalence in donkeys in Mexico. Meat from donkey is consumed by humans in Mexico and also exported to other countries. We sought to determine the presence of antibodies against T. gondii in 239 domestic donkeys (Equus asinus) for slaughter in Durango, Mexico using the modified agglutination test (MAT). Donkeys were sampled in four premises (trade centers) where donkeys were gather for shipment to abattoirs in other Mexican states.
Antibodies to T. gondii were found in 26 (10.9%) of 239 donkeys, with MAT titers of 1:25 in 7, 1:50 in 11, 1:100 in 6, and 1:200 in 2. Seropositive donkeys were found in three (75%) of the four gathering premises studied. Seroprevalence in donkeys varied from 0% to 23.1% among gathering premises. The seroprevalence of T. gondii infection was comparable among donkeys regardless their age, sex or health status. Seropositivity to T. gondii was found in donkeys between 1 to 12 years old. Multivariate analysis showed that seropositivity to T. gondii was associated with the gathering premises (OR = 1.58; 95% CI: 1.11-2.24; P = 0.009).
This is the first report of T. gondii infection in donkeys in Mexico. Results indicate that consumption of undercooked or raw meat from T. gondii-infected donkeys is potentially a source of T. gondii infection for humans.
PMCID: PMC4301930  PMID: 25595816
Toxoplasma gondii; Seroprevalence; Donkeys; Cross-sectional study; Mexico
34.  Epidemiology of Theileria bicornis among black and white rhinoceros metapopulation in Kenya 
A huge effort in rhinoceros conservation has focused on poaching and habitat loss as factors leading to the dramatic declines in the endangered eastern black rhinoceros (Diceros bicornis michaeli) and the southern white rhinoceros (Ceratotherium simum simum). Nevertheless, the role disease and parasite infections play in the mortality of protected populations has largely received limited attention. Infections with piroplasmosis caused by Babesia bicornis and Theileria bicornis has been shown to be fatal especially in small and isolated populations in Tanzania and South Africa. However, the occurrence and epidemiology of these parasites in Kenyan rhinoceros is not known.
Utilizing 18S rRNA gene as genetic marker to detect rhinoceros infection with Babesia and Theileria, we examined blood samples collected from seven rhinoceros populations consisting of 114 individuals of black and white rhinoceros. The goal was to determine the prevalence in Kenyan populations, and to assess the association of Babesia and Theileria infection with host species, age, sex, location, season and population mix (only black rhinoceros comparing to black and white rhinoceros populations). We did not detect any infection with Babesia in the sequenced samples, while the prevalence of T. bicornis in the Kenyan rhinoceros population was 49.12% (56/114). White rhinoceros had significantly higher prevalence of infection (66%) compared to black rhinoceros (43%). The infection of rhinoceros with Theileria was not associated with animal age, sex or location. The risk of infection with Theileria was not higher in mixed species populations compared to populations of pure black rhinoceros.
In the rhinoceros studied, we did not detect the presence of Babesia bicornis, while Theileria bicornis was found to have a 49.12% prevalence with white rhinoceros showing a higher prevalence (66%) comparing with black rhinoceros (43%). Other factors such as age, sex, location, and population mix were not found to play a significant role.
PMCID: PMC4328958  PMID: 25604678
Ixodid; Ticks; Piroplasms; Diceros bicornis michaeli; Ceratotherium simum simum
35.  An association between milk and slime increases biofilm production by bovine Staphylococcus aureus 
Staphylococcus aureus is associated with chronic mastitis in cattle, and disease manifestation is usually refractory to antibiotic therapy. Biofilm production is a key element of S. aureus pathogenesis and may contribute to the treatment failure that is consistently reported by veterinarians. Minas Gerais State is the largest milk-producing state in Brazil, and the characterization of bacterial isolates is an important aspect of disease control for dairy farmers. Here, we investigated the potential of S. aureus isolated from bovine mastitis to produce slime and biofilm in a skim-milk medium and classified the isolates according to their agr type.
Slime was detected using the Congo Red agar (CRA) test in 35.18% (19/54) of the strains; however, 87.04% (47/54) of the strains were considered biofilm-positive based on crystal violet staining. Compared to TSB supplemented with 0.25% glucose, skim milk significantly increased the production of biofilm, but this effect was only observed in slime-producing strains. The bacteria belonged to agr groups I (12/54), II (34/54), III (6/54), and IV (2/54), and bacteria in agr group III were found to be stronger biofilm producers than those in groups I and II. Again, milk had a significant influence only on slime-positive agr I and II isolates, revealing an association between milk and slime.
The present study demonstrated that skim-milk medium and slime production are two factors that together influence biofilm formation by bovine strains of S. aureus. A predominance of bacteria belonging to agr group II was observed, and bacteria from agr group III showed the highest proportion of biofilm producers. The majority of bacteria characterized in this study formed biofilm in milk, which suggests that biofilm formation has an important role in the virulence of S. aureus isolated from bovine intramammary infections.
PMCID: PMC4311514  PMID: 25591667
Staphylococcus aureus, Biofilm, Slime, Skim-milk medium; agr type
36.  Development of a nanoparticle-assisted PCR (nanoPCR) assay for detection of mink enteritis virus (MEV) and genetic characterization of the NS1 gene in four Chinese MEV strains 
Mink enteritis virus (MEV) causes mink viral enteritis, an acute and highly contagious disease whose symptoms include violent diarrhea, and which is characterized by high morbidity and mortality. Nanoparticle-assisted polymerase chain reaction (nanoPCR) is a recently developed technique for the rapid detection of bacterial and viral DNA. Here we describe a novel nanoPCR assay for the clinical detection and epidemiological characterization of MEV.
This assay is based upon primers specific for the conserved region of the MEV NS1 gene, which encodes nonstructural protein 1. Under optimized conditions, the MEV nanoPCR assay had a detection limit of 8.75 × 101 copies recombinant plasmids per reaction, compared with 8.75 × 103 copies for conventional PCR analysis. Moreover, of 246 clinical mink samples collected from five provinces in North-Eastern China, 50.8% were scored MEV positive by our nanoPCR assay, compared with 32.5% for conventional PCR. Furthermore no cross reactivity was observed for the nanoPCR assay with respect to related viruses, including canine distemper virus (CDV) and Aleutian mink disease parvovirus (AMDV). Phylogenetic analysis of four Chinese wild type MEV isolates using the nanoPCR assay indicated that they belonged to a small MEV clade, named “China type”, in the MEV/FPLV cluster, and were closely clustered in the same location.
Our results indicate that the MEV China type clade is currently circulating in domestic minks in China. We anticipate that the nanoPCR assay we have described here will be useful for the detection and epidemiological and pathological characterization of MEV.
Electronic supplementary material
The online version of this article (doi:10.1186/s12917-014-0312-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4300173  PMID: 25582057
Nanoparticle-assisted PCR; Mink enteritis virus; Nonstructural protein 1 gene; Genetic characterization; China type
37.  Development of learning objectives for neurology in a veterinary curriculum: part I: undergraduates 
With an increasing caseload of veterinary neurology patients in first opinion practice, there is a requirement to establish relevant learning objectives for veterinary neurology encompassing knowledge, skills and attitudes for veterinary undergraduate students in Europe. With help of experts in veterinary neurology from the European College of Veterinary Neurology (ECVN) and the European Society of Veterinary Neurology (ESVN) a survey of veterinary neurologic learning objectives using a modified Delphi method was conducted. The first phase comprised the development of a draft job description and learning objectives by a working group established by the ECVN. In the second phase, a quantitative questionnaire (multiple choice, Likert scale and free text) covering 140 learning objectives and subdivided into 8 categories was sent to 341 ESVN and ECVN members and a return rate of 62% (n = 213/341) was achieved.
Of these 140 learning objectives ECVN Diplomates and ESVN members considered 42 (30%) objectives as not necessary for standard clinical veterinary neurology training, 94 (67%) were graded to be learned at a beginner level and 4 (3%) at an advanced level. The following objectives were interpreted as the most important day one skills: interpret laboratory tests, perform a neurological examination and establish a neuroanatomical localization. In this survey the three most important diseases of the central nervous system included epilepsy, intervertebral disc disease and inflammatory diseases. The three most important diseases of the peripheral nervous system included polyradiculoneuritis, myasthenia gravis and toxic neuropathies.
The results of this study should help to reform the veterinary curriculum regarding neurology and may reduce the phenomenon of “Neurophobia”.
Electronic supplementary material
The online version of this article (doi:10.1186/s12917-014-0315-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4300725  PMID: 25582136
Veterinary education; Curriculum; Learning objectives; Neurology; Undergraduate; ECVN; ESVN; Europe
38.  A rare homozygous MFSD8 single-base-pair deletion and frameshift in the whole genome sequence of a Chinese Crested dog with neuronal ceroid lipofuscinosis 
BMC Veterinary Research  2015;10(1):960.
The neuronal ceroid lipofuscinoses are heritable lysosomal storage diseases characterized by progressive neurological impairment and the accumulation of autofluorescent storage granules in neurons and other cell types. Various forms of human neuronal ceroid lipofuscinosis have been attributed to mutations in at least 13 different genes. So far, mutations in the canine orthologs of 7 of these genes have been identified in DNA from dogs with neuronal ceroid lipofuscinosis. The identification of new causal mutations could lead to the establishment of canine models to investigate the pathogenesis of the corresponding human neuronal ceroid lipofuscinoses and to evaluate and optimize therapeutic interventions for these fatal human diseases.
Case presentation
We obtained blood and formalin-fixed paraffin-embedded brain sections from a rescue dog that was reported to be a young adult Chinese Crested. The dog was euthanized at approximately 19 months of age as a consequence of progressive neurological decline that included blindness, anxiety, and cognitive impairment. A diagnosis of neuronal ceroid lipofuscinosis was made based on neurological signs, magnetic resonance imaging of the brain, and fluorescence microscopic and electron microscopic examination of brain sections. We isolated DNA from the blood and used it to generate a whole genome sequence with 33-fold average coverage. Among the 7.2 million potential sequence variants revealed by aligning the sequence reads to the canine genome reference sequence was a homozygous single base pair deletion in the canine ortholog of one of 13 known human NCL genes: MFSD8:c.843delT. MFSD8:c.843delT is predicted to cause a frame shift and premature stop codon resulting in a truncated protein, MFSD8:p.F282Lfs13*, missing its 239 C-terminal amino acids. The MFSD8:c.843delT allele is absent from the whole genome sequences of 101 healthy canids or dogs with other diseases. The genotyping of archived DNA from 1478 Chinese Cresteds did not identify any additional MFSD8:c.843delT homozygotes and found only one heterozygote.
We conclude that the neurodegenerative disease of the Chinese Crested rescue dog was neuronal ceroid lipofuscinosis and that homozygosity for the MFSD8:c.843delT sequence variant was very likely to be the molecular-genetic cause of the disease.
Electronic supplementary material
The online version of this article (doi:10.1186/s12917-014-0181-z) contains supplementary material, which is available to authorized users.
PMCID: PMC4298050  PMID: 25551667
MFSD8; Neuronal ceroid lipofuscinosis; Chinese Crested; Whole genome sequence
39.  Influence of repeated anaesthesia on physiological parameters in male Wistar rats: a telemetric study about isoflurane, ketamine-xylazine and a combination of medetomidine, midazolam and fentanyl 
BMC Veterinary Research  2014;10(1):310.
This study evaluated the influence of repeated anaesthesia using isoflurane (ISO, 2–3 Vol%), ketamine-xylazine (KX, 100 mg·kg−1 + 5 mg·kg−1, i.m.) or a combination of medetomidine-midazolam-fentanyl (MMF, 0.15 mg·kg−1 + 2.0 mg·kg−1 + 0.005 mg·kg−1, i.m.) on heart rate (HR), arterial blood pressure (BP), body temperature (BT), duration of anaesthetic intervals and body weight (BW) in Wistar rats. Rats were instrumented with a telemetric system for the measurement of systolic, diastolic and mean arterial pressure (SAP, DAP, MAP), pulse pressure (PP), HR and BT during induction, maintenance and recovery of anaesthesia. Each anaesthesia was performed six times within three weeks. KX was not antagonized, but ISO delivery was terminated 40 minutes after induction and MMF was reversed with atipamezole-flumazenil-naloxone (AFN, 0.75 mg·kg−1 + 0.2 mg·kg−1 + 0.12 mg·kg−1, s.c.).
With repeated anaesthesia, ISO showed a decrease of HR and BP. A significant decrease of PP could be observed with repeated anaesthesia using MMF. HR and BP were not affected by repeated KX anaesthesia, but we noted a reduction of sleeping time and BW. Neither MMF nor ISO showed significant differences in the duration of anaesthetic intervals and BW. With KX we observed tissue necrosis at the injection site and surgical tolerance was not achieved in 25% of the anaesthesias performed.
HR, BP values, BT, duration of anaesthetic intervals and BW were affected differently by repeated anaesthesia performed with ISO, KX or MMF. ISO produced a reproducible anaesthesia, thereby being suitable for repeated use, but with a decrease of HR and BP throughout the six anaesthesias. The use of ISO in cases where these parameters should be unaffected is therefore not advised. The inability to produce a surgical tolerance, the reduction of sleeping time and BW, as well as the tissue necrosis are significant contraindications for a repeated use of KX. Only mild changes of BP were found with repeated MMF anaesthesia, so it seems suitable for serial use, unless the high BP and the low HR during the surgical plane of anaesthesia are undesirable for a special procedure.
PMCID: PMC4301083  PMID: 25551200
Rat; Repeated anaesthesia; Isoflurane; Ketamine-xylazine; Medetomidine-midazolam-fentanyl; Telemetry; Heart rate; Blood pressure; Body temperature; Body weight
40.  Enhanced Wnt/β-catenin and Notch signalling in the activated canine hepatic progenitor cell niche 
BMC Veterinary Research  2014;10(1):309.
The liver has a large regenerative capacity. Hepatocytes can replicate and regenerate a diseased liver. However, as is the case in severe liver diseases, this replication may become insufficient or exhausted and hepatic progenitor cells (HPCs) can be activated in an attempt to restore liver function. Due to their bi-potent differentiation capacity, these HPCs have great potential for regenerative approaches yet over-activation does pose potential health risks. Therefore the mechanisms leading to activation must be elucidated prior to safe implementation in the veterinary clinic. Wnt/β-catenin and Notch signalling have been implicated in the activation of HPCs in mouse models and in humans. Here we assessed the involvement in canine HPC activation. Gene-expression profiles were derived from laser microdissected HPC niches from lobular dissecting hepatitis (LDH) and normal liver tissue, with a focus on Wnt/β-catenin and Notch signalling. Immunohistochemical and immunofluorescent studies were combined to assess the role of the pathways in HPCs during LDH.
Gene-expression confirmed higher expression of Wnt/β-catenin and Notch pathway components and target genes in activated HPC niches in diseased liver compared to quiescent HPC niches from normal liver. Immunofluorescence confirmed the activation of these pathways in the HPCs during disease. Immunohistochemistry showed proliferating HPCs during LDH, and double immunofluorescence showed downregulation of Wnt/β-catenin and Notch in differentiating HPCs. Vimentin, a mesenchymal marker, was expressed on a subset of undifferentiated HPCs.
Together these studies clearly revealed that both Wnt/β-catenin and Notch signalling pathways are enhanced in undifferentiated, proliferating and potentially migrating HPCs during severe progressive canine liver disease (LDH).
PMCID: PMC4302101  PMID: 25551829
Laser microdissection; Hepatic progenitor cells; Dog; Wnt/β-catenin; Notch signalling
41.  Ultrasound imaging of the anterior section of the eye of five different snake species 
BMC Veterinary Research  2014;10(1):313.
Nineteen clinically normal snakes: six ball pythons (Python regius), six Burmese pythons (Python bivittatus), one Children’s python (Antaresia childreni), four Amazon tree boas (Corallus hortulanus), and two Malagasy ground boas (Acrantophis madagascariensis) were subjected to ultrasound imaging with 21 MHz (ball python) and 50 MHz (ball python, Burmese python, Children’s python, Amazon tree boa, Malagasy ground boa) transducers in order to measure the different structures of the anterior segment in clinically normal snake eyes with the aim to review baseline values for clinically important ophthalmic structures. The ultrasonographic measurements included horizontal spectacle diameter, spectacle thickness, depth of sub-spectacular space and corneal thickness. For comparative purposes, a formalin-fixed head of a Burmese python was subjected to micro computed tomography.
In all snakes, the spectacle was thinner than the cornea. There was significant difference in spectacle diameter, and spectacle and corneal thickness between the Amazon tree boa and the Burmese and ball pythons. There was no difference in the depth of the sub-spectacular space. The results obtained in the Burmese python with the 50 MHz transducer were similar to the results obtained with micro computed tomography. Images acquired with the 21 MHz transducer included artifacts which may be misinterpreted as ocular structures.
Our measurements of the structures in the anterior segment of the eye can serve as orientative values for snakes examined for ocular diseases. In addition, we demonstrated that using a high frequency transducer minimizes the risk of misinterpreting artifacts as ocular structures.
Electronic supplementary material
The online version of this article (doi:10.1186/s12917-014-0313-5) contains supplementary material, which is available to authorized users.
PMCID: PMC4302111  PMID: 25547871
Snake; Spectacle; Ultrasound
42.  Comprehensive report of an Enterococcus cecorum infection in a broiler flock in Northern Germany 
BMC Veterinary Research  2014;10(1):311.
Enterococcus cecorum is considered as an emerging pathogen in poultry and can cause substantial losses in broiler flocks. Femoral head necrosis and spondylitis were described as the main pathological changes in infected chickens. Nevertheless, little is known about the pathogenesis of Enterococcus cecorum infection in broilers. This report shows for the first time the whole course of disease over an entire growing period including repeated necropsies and subsequent microbiological investigations.
Case presentation
In a flock of 18200 broilers, a decrease in flock uniformity was detected from 14 days post hatch onwards with affected chickens showing lameness and an increase in flock mortality up to 7.22% at day 33 post hatch. In the first 3 weeks post hatch, pericarditis and hepatitis were found as the main pathological changes in 27.6% and 9.8% of the examined broilers respectively. Femoral head necrosis and vertebral osteomyelitis were detected in the last week of the growing period with 10.3% and 2.3% respectively. Heart, liver, spleen, yolk sac and vertebral column of 59 broilers with pathological changes were subjected to bacteriological analysis. Enterococcus cecorum was isolated from 23 birds (39%), the first broiler was already positive at day 3 post hatch in the yolk sac. Additionally, 9.75% of the broilers were rejected at the slaughterhouse primarily because of pathological changes. The investigated broiler cycle had by far the best footpad score compared to 7 cycles before and 4 cycles after the Enterococcus cecorum infection at the same farm.
Bacteraemia and generalized infection appear to be important steps in the pathogenesis of Enterococcus cecorum infection in broilers. Furthermore, this disease causes economic losses for the farmer not only due to an increase in flock mortality, but probably also through substantially higher condemnation rates at the slaughterhouse. It was speculated that the broilers were infected via the respiratory tract as this flock had lower footpad scores likely the result of drier litter. The latter may have led to higher dust concentrations and thus airborne Enterococcus cecorum.
PMCID: PMC4297365  PMID: 25539747
Gallus gallus domesticus; Chicken; Broiler; Infection; Septicemia; Osteomyelitis; Pericarditis; Hepatitis; Enterococcus cecorum; Pathogenesis; Route of infection; Condemnation rate
43.  The deletion of an extra six nucleotides in the 5′ -untranslated region of the nucleoprotein gene of Newcastle disease virus NA-1 decreases virulence 
BMC Veterinary Research  2014;10(1):964.
The virulent Newcastle disease virus (NDV) strain NA-1 (genotype VII) was isolated from an epizootic involving a flock of geese in Jilin Province, Northeast China, in 1999. Compared with the classical NDV strains, which have a genome size of 15,186 bp, the more recently isolated NDV strains, including that involved in the goose outbreak, have an extra six nucleotides in the 5′-untranslated region (UTR) of the nucleoprotein (NP) gene. This extra sequence, TCCCAC, is highly conserved and has been found in multiple NDV strains, including ZJ-1, WF00G, JSG0210, and NA-1. In the current study, an infectious clone from strain NA-1 was isolated and designated rNA-1. Subsequently, strain rNA-1 was mutated to delete the six-nucleotide insertion, producing strain rNA-1(−). Virulence of the recombinant virus was then assayed in chickens and geese.
The recombinant virus rNA-1(−) showed similar biological characteristics to the parental NA-1 strain in DF-1 chicken fibroblast cells. However, the virulence of rNA-1(−) in 9-day-old embryonated chicken eggs and 1-day-old specific pathogen-free (SPF) chickens was decreased compared with the rNA-1 control. Furthermore, the virulence of the recombinant strain was slightly decreased in 1-day-old SPF chickens when compared with that in 1-day-old geese.
Following deletion of six nucleotides in the 5′-UTR of the NP gene of NDV strain NA-1, the virulence of the rNA-1(−) recombinant strain was decreased in both chickens and geese. However, rNA-1(−) was more virulent in chickens than geese, as seen by the higher intracerebral pathogenicity index value.
PMCID: PMC4297397  PMID: 25528581
Goose; Newcastle disease virus; Reverse genetics
44.  Physiological and analgesic effects of continuous-rate infusion of morphine, butorphanol, tramadol or methadone in horses with lipopolysaccharide (LPS)-induced carpal synovitis 
BMC Veterinary Research  2014;10(1):966.
Continuous-rate infusion (CRI) of drugs results in more stable plasma drug concentrations than administration of intermittent boluses, thus providing greater stability of physiological parameters. The aim of this study was to evaluate the physiologic and analgesic effects of the administration of morphine, butorphanol, tramadol or methadone by CRI in horses with induced synovitis of the radiocarpal joint.
Increased values of cardiorespiratory parameters and body temperature were observed in all groups after initiation of opioid administration, and these increases were sustained throughout the CRI period. Morphine, butorphanol and methadone each caused a reduction in gut sounds, and this effect was greatest in animals that received morphine. Administration of morphine or methadone reduced the degree of lameness after the end of intravenous infusion. Administration of tramadol did not alter the degree of lameness in the animals.
CRI of morphine or methadone, but not butorphanol or tramadol, provided analgesia in horses with carpal synovitis. All of these opioids increased cardiovascular and respiratory parameters and reduced gut sounds during CRI.
PMCID: PMC4297404  PMID: 25528353
Opioids; Analgesia; Inflammatory pain
45.  First molecular detection and characterization of herpesvirus and poxvirus in a Pacific walrus (Odobenus rosmarus divergens) 
BMC Veterinary Research  2014;10(1):968.
Herpesvirus and poxvirus can infect a wide range of species: herpesvirus genetic material has been detected and amplified in five species of the superfamily Pinnipedia; poxvirus genetic material, in eight species of Pinnipedia. To date, however, genetic material of these viruses has not been detected in walrus (Odobenus rosmarus), another marine mammal of the Pinnipedia clade, even though anti-herpesvirus antibodies have been detected in these animals.
Case presentation
In February 2013, a 9-year-old healthy captive female Pacific walrus died unexpectedly at L’Oceanografic (Valencia, Spain). Herpesvirus was detected in pharyngeal tonsil tissue by PCR. Phylogenetic analysis revealed that the virus belongs to the subfamily Gammaherpesvirinae. Poxvirus was also detected by PCR in skin, pre-scapular and tracheobronchial lymph nodes and tonsils. Gross lesions were not detected in any tissue, but histopathological analyses of pharyngeal tonsils and lymph nodes revealed remarkable lymphoid depletion and lymphocytolysis. Similar histopathological lesions have been previously described in bovine calves infected with an alphaherpesvirus, and in northern elephant seals infected with a gammaherpesvirus that is closely related to the herpesvirus found in this case. Intracytoplasmic eosinophilic inclusion bodies, consistent with poxviral infection, were also observed in the epithelium of the tonsilar mucosa.
To our knowledge, this is the first molecular identification of herpesvirus and poxvirus in a walrus. Neither virus was likely to have contributed directly to the death of our animal.
PMCID: PMC4299291  PMID: 25527906
Herpesvirus; Poxvirus; Walrus; Pinniped
46.  Cross-sectional study of Schmallenberg virus seroprevalence in wild ruminants in Poland at the end of the vector season of 2013 
BMC Veterinary Research  2014;10(1):967.
In view of recurrent Schmallenberg virus (SBV) infections all over Europe between 2011 and 2013, a lively scientific debate over the importance of the sylvatic transmission cycle of the virus has emerged. The study presents results of serosurvey which included wild ruminants representing species of red deer (Cervus elaphus), roe deer (Capreolus capreolus), European bison (Bison bonasus), fallow deer (Dama dama), mouflon (Ovis orientalis musimon) hunted or immobilized at 34 different locations of Poland in the autumn/winter 2013.
Out of 580 sera, 145 (25%) were considered positive for SBV antibodies. The overall SBV seroprevalence calculated using district probability weights was estimated at 27.7% (95% CI: 24.0-31.4). The seroprevalences at the district level varied between 0 and 80.0% (95% CI: 24.5-135.0%) with the mean within-district prevalence of 24.0% (95% CI: 16.5-31.4). Significantly higher seroprevalence was observed in animals from the Eastern provinces (36.6%) compared to the Western provinces (22.8%). SBV infection impact varied significantly between different species (higher SBV seroprevalence in larger species such as European bison), population type (free-ranging; captive), age, body weight, percent of the district forest area, part of Poland, and the densities of wild and domestic ruminants at the district and province level. Using statistical multivariable logistic model, population type, age, part of Poland and domestic ruminant density were identified as the main risk factors for SBV infection in wild ruminants in Poland.
SBV seroprevalence in wild ruminants, similarly to the epizootic situation in domestic ruminants in the country, varied significantly between districts and provinces. Association between SBV seropositivity, species, animal body weight and age group expressed by a higher prevalence in larger ruminants may be explained by more frequent exposure to midge-vector bites of the latter, however it might also be related to the different species susceptibility to SBV infection. The positive effect of higher domestic ruminant density on the risk of SBV infection in wildlife and lower SBV seroprevalences in the latter suggested that the sylvatic cycle of SBV transmission is an effect of the pathogen spillover from the domestic animals.
PMCID: PMC4299547  PMID: 25528665
Schmallenberg virus; Wild ruminants; Seroprevalence; Risk factors
47.  Clinical skills of veterinary students – a cross-sectional study of the self-concept and exposure to skills training in Hannover, Germany 
BMC Veterinary Research  2014;10(1):969.
Students of veterinary medicine should achieve basic professional competences required to practise their profession. A main focus of veterinary education is on developing clinical skills.
The present study used the guidelines of the “Day-One Skills” list of European Association of Establishments for Veterinary Education (EAEVE) to create an online questionnaire for assessing the skills acquired by students at the University of Veterinary Medicine Hannover (TiHo). The theoretical and practical veterinary knowledge levels of the students and postgraduates are determined and compared.
In two batches, 607 people responded (response batch 1, 23.78%; response batch 2, 23.83%). From 49 defined skills, 28 are actually practised during training at the university and 21 activities are known only theoretically. Furthermore, the students showed great willingness to use simulators and models in a clinical skills lab.
The results of this survey highlight that the opening of a clinical skills lab at the University of Veterinary Medicine Hannover and its incorporation into the study programme are ideal tools to promote practical competences and foster the motivation to learn.
Electronic supplementary material
The online version of this article (doi:10.1186/s12917-014-0302-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4300046  PMID: 25528469
Clinical skills lab; Clinical skills; Questionnaire; Cross-sectional study; Veterinary teaching
48.  Corynebacterium pseudotuberculosis cp09 mutant and cp40 recombinant protein partially protect mice against caseous lymphadenitis 
BMC Veterinary Research  2014;10(1):965.
Caseous lymphadenitis (CLA) is an infectious disease that affects small ruminants and is caused by Corynebacterium pseudotuberculosis. This disease is responsible for high economic losses due to condemnation and trim of infected carcasses, decreased leather and wool yield, loss of sales of breeding stock and deaths from internal involvement. Treatment is costly and ineffective; the most cost-effective strategy is timely immunisation. Various vaccine strategies have been tested, and recombinant vaccines are a promising alternative. Thus, in this study, different vaccine formulations using a recombinant protein (rCP40) and the CP09 live recombinant strain were evaluated. Five groups of 10 mice each were immunised with saline (G1), rCP40 (G2), CP09 (G3), a combination of CP09 and rCP40 (G4) and a heterologous prime-boost strategy (G5). Mice received two immunisations within 15 days. On day 30 after primary immunisation, all groups were challenged with a C. pseudotuberculosis virulent strain. Mice were monitored and mortality was recorded for 30 days after challenge.
The G2, G4 and G5 groups showed high levels of IgG1 and IgG2a; G2 presented significant IgG2a production after virulent challenge in the absence of IgG1 and IgG3 induction. Thirty days after challenge, the mice survival rates were 20 (G1), 90 (G2), 50 (G3), 70 (G4) and 60% (G5).
rCP40 is a promising target in the development of vaccines against caseous lymphadenitis.
PMCID: PMC4297461  PMID: 25527190
Caseous lymphadenitis; Corynebacterium pseudotuberculosis; Recombinant vaccines; Live attenuated vaccines
49.  Clinical efficacy of 9-oxo-10, 11-dehydroageraphorone extracted from Eupatorium adenophorum against Psoroptes cuniculi in rabbits 
BMC Veterinary Research  2014;10(1):970.
Animal acariasis is one of the important veterinary skin diseases. Chemical drugs have been widely used to treat and control this kind of disease. But many chemicals control could increase resistance in target species, toxicity and environmental hazards. We found that the 9-oxo-10, 11-dehydroageraphorone (euptox A) extracted from E. adenophorum has strong toxicity against P. cuniculi in vitro, but the in vivo acaricidal actions of euptox A have yet to be investigated.
A 14-day experiment was performed using rabbits that were naturally infested with P. cuniculi on a farm. Rabbits were randomly divided into five groups; animals in groups A, B and C were treated in each ear topically with 4.0 ml of 2.0 and 1.0 g/L (w/v) euptox A, respectively. Animals in groups D and E were treated with ivermectin (by injection; positive controls) and glycerol with water only (by embrocation; negative controls), respectively. Each rabbit was treated twice with separate treatments on days 0 and 7. Rabbits were observed daily and detailed examinations were performed on days 0, 7 and 14, to inspect the presence or absence of mites and scabs/crusts. Seven days after the initial treatment, the mean clinical scores (presence of scabs/crusts) decreased from 3.48, 3.37, 3.43 and 3.45 to 0.37, 0.42, 0.78 and 0.38 in the ears of animals in groups A, B , C and D, respectively, which were similar to the observations recorded in the positive control rabbits. However, the clinical score for negative control rabbits did not increase significantly (P > 0.05) during the experiment, and this changed from 3.32 to 3.37 in the ears, and there were no significant differences in clinical efficacy between left and right ears. After two treatments (0 and 7 d), the rabbits in groups A, B, C and D had recovered completely 14 days after the last treatment and no recurrences of infection were observed.
These results indicate that euptox A was potent compounds for the effective control of animal P. cuniculi in vivo.
PMCID: PMC4308076  PMID: 25527276
Euptox A; Eupatorium adenophorum; Psoroptes cuniculi; Acaricidal; Clinical efficacy
50.  Treatment with embryonic stem-like cells into osteochondral defects in sheep femoral condyles 
BMC Veterinary Research  2014;10(1):301.
Articular cartilage has poor intrinsic capacity for regeneration because of its avascularity and very slow cellular turnover. Defects deriving from trauma or joint disease tend to be repaired with fibrocartilage rather than hyaline cartilage. Consequent degenerative processes are related to the width and depth of the defect. Since mesenchymal stem cells (MSCs) deriving from patients affected by osteoarthritis have a lower proliferative and chondrogenic activity, the systemic or local delivery of heterologous cells may enhance regeneration or inhibit the progressive loss of joint tissue. Embryonic stem cells (ESCs) are very promising, since they can self-renew for prolonged periods without differentiation and can differentiate into tissues from all the 3 germ layers. To date only a few experiments have used ESCs for the study of the cartilage regeneration in animal models and most of them used laboratory animals. Sheep, due to their anatomical, physiological and immunological similarity to humans, represent a valid model for translational studies. This experiment aimed to evaluate if the local delivery of male sheep embryonic stem-like (ES-like) cells into osteochondral defects in the femoral condyles of adult sheep can enhance the regeneration of articular cartilage. Twenty-two ewes were divided into 5 groups (1, 2, 6, 12 and 24 months after surgery). Newly formed tissue was evaluated by macroscopic, histological, immunohistochemical (collagen type II) and fluorescent in situ hybridization (FISH) assays.
Regenerated tissue was ultimately evaluated on 17 sheep. Samples engrafted with ES-like cells had significantly better histologic evidence of regeneration with respect to empty defects, used as controls, at all time periods.
Histological assessments demonstrated that the local delivery of ES-like cells into osteochondral defects in sheep femoral condyles enhances the regeneration of the articular hyaline cartilage, without signs of immune rejection or teratoma for 24 months after engraftment.
Electronic supplementary material
The online version of this article (doi:10.1186/s12917-014-0301-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4297431  PMID: 25523522
Articular cartilage; Embryonic stem-like cell; Fluorescent in situ hybridization; Osteochondral defect; Sheep

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