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1.  Reduced birth weight, cleft palate and preputial abnormalities in a cloned dog 
The aim of the present study was to report a novel developmental abnormality in a cloned dog. A fibroblast cell line was established from an 8-year-old male German shepherd dog. In vivo matured oocytes were retrieved from a large breed dog, and the nucleus was removed from each oocyte. A donor cell was injected into an enucleated oocyte, and the oocyte-cell couplet was fused electrically. After chemical activation, the resulting embryos were transferred into a naturally estrus-synchronized recipient dog, and two cloned pups were delivered by Cesarean section 60 days later. One cloned pup (Clone 1) was healthy, but the other (Clone 2) had a birth weight of only 320 g and cleft palate, failure of preputial closure at the ventral distal part, and persistent penile frenulum. Clone 2 was raised by stomach feeding until Day 40 after birth, where palatoplasty was performed. The abnormalities in external genitalia in Clone 2 resulted in persistent penile extrusion that was surgically corrected. This complex developmental abnormality has not been reported in dogs previously.
doi:10.1186/1751-0147-56-18
PMCID: PMC3984017  PMID: 24669802
Developmental abnormality; Cleft palate; Penile frenulum; Preputial closure; Persistent penile extrusion; Cloning; Dog
2.  Tetralogy of Fallot and atrial septal defect in a white Bengal Tiger cub (Panthera tigris tigris) 
A 3-week-old female white Bengal Tiger cub (Panthera tigris tigris) presented with acute onset tachypnoea, cyanosis and hypothermia. The cub was severely hypoxaemic with a mixed acid–base disturbance. Echocardiography revealed severe pulmonic stenosis, right ventricular hypertrophy, high membranous ventricular septal defect and an overriding aorta. Additionally, an atrial septal defect was found on necropsy, resulting in the final diagnosis of Tetralogy of Fallot with an atrial septal defect (a subclass of Pentalogy of Fallot). This report is the first to encompass arterial blood gas analysis, thoracic radiographs, echocardiography and necropsy findings in a white Bengal Tiger cub diagnosed with Tetralogy of Fallot with an atrial septal defect.
doi:10.1186/1751-0147-56-12
PMCID: PMC3984729  PMID: 24594084
Pentalogy; Pulmonic stenosis; Echocardiography; Necropsy
3.  Exercise induced hypercoagulability, increased von Willebrand factor and decreased thyroid hormone concentrations in sled dogs 
Background
Sled dogs performing endurance races have been reported to have a high incidence of gastric erosions or ulcerations and an increased risk of gastro intestinal bleeding leading to death in some cases. In addition, these dogs also become hypothyroid during training and exercise. Canine hypothyroidism has been shown to correlate with decreased von Willebrand factor antigen and potentially increased bleeding tendency. Whether increased gastro intestinal bleeding risk is exacerbated due to changes in the hemostatic balance is unknown. The aim of this study was to investigate the hemostatic balance in sled dogs before and after exercise and in addition evaluate any correlation to thyroid status. Twenty sled dogs have been assessed in untrained and trained condition and immediately after exercise. The first sample was collected in the autumn following a resting period, and subsequently the dogs were exposed to increased intensity of training. After four months the peak of physical condition was reached and a 68 km long sled pulling exercise was performed. Samples were collected before and immediately after the exercise. Evaluated parameters were: plasma thromboelastographic (TEG) R, SP, α and MA, activated partial thromboplastin time (aPTT), prothrombin time (PT), fibrinogen, von Willebrand factor (vWf), D-dimer, platelet number, thyroid hormones, hematocrit and C-reactive protein (CRP).
Results
Exercise induced an overall hypercoagulable state characterized by significant decreases of TEG R and SP and an increase of α, increased concentrations of plasma vWf and decreased aPTT. In addition, a proinflammatory status was seen by a significant increase of serum CRP concentrations. Thyroid status was confirmed to be hypothyroid as training and exercise induced significant decrease of thyroxin (T4), free thyroxin (fT4) and thyroxin stimulating hormone (TSH) concentrations. Fibrinogen decreased significantly and PT increased. The training-induced changes showed correlation between T4, fT4 and aPTT and correlation between TSH and fibrinogen. Exercise-induced changes showed correlation between T4 and PT.
Conclusions
Exercise was associated with a hypercoagulable state and an increase of vWf concentration in this group of sled dogs. Decreased thyroid hormone concentrations after training and exercise were confirmed, but were associated with increased and not decreased vWf in this group of sled dogs.
doi:10.1186/1751-0147-56-11
PMCID: PMC3922025  PMID: 24507241
Canine; Exercise; Gastric lesions; Hemostasis; Sled dogs; Thromboelastography; Thyroid hormones
4.  Analysis of blood gases, serum fat and serum protein: a new approach to estimate survival chances of stranded Harbor seal (Phoca vitulina) pups from the German North Sea 
Background
Facing numerous challenges, such as illness, storms or human disturbance, some harbor seal (Phoca vitulina) pups lose contact to their dams and are found abandoned along the North Sea coast. In Schleswig-Holstein, pups with the prospect of surviving rehabilitation are admitted to the Seal Center Friedrichskoog. Despite elaborate clinical health assessments on admission, including differential hematology, in 2010, 17% of 108 admitted pups did not survive the first 20 days. The death rate during the years 2006 and 2009 varied between 9 and 19%. To broaden the spectrum of variables which could be predictive for survival, blood gas and serum analyses were performed for 99 pups using venous blood. Variables included total CO2, pH, partial CO2, HCO3–, base excess and anion gap as well as glucose, urea nitrogen, sodium, potassium and chloride. Moreover, total serum protein and fat (triglyceride) concentrations were measured for all pups on admission.
Results
Repeated measurements of 12 randomly selected individuals revealed a significant (p = 0.002) positive influence of time in rehabilitation on triglyceride concentrations. This trend probably shows the improvement of the pups’ nutritional status as a consequence of the shift from milk replacer formula to fish. No such positive influence was detected for total protein concentrations though. Hematologic values, including blood gases, were not predictive for survival.
Conclusions
For the first time blood gas values are reported in this study for a large sample size (N = 99) of seal pups (regardless of their health status). The ranges and medians calculated from the data can serve as a stepping stone towards the establishment of reference values for neonate harbor seals. However, future investigations on the development of blood gases in harbor seals with different health conditions and ages over time are necessary to allow for a better understanding of acid–base regulation in harbor seals.
doi:10.1186/1751-0147-56-10
PMCID: PMC3930289  PMID: 24490584
Blood gas analysis; Serum fat (triglycerides); Serum protein; Harbor seal pups; Rehabilitation; Phoca vitulina
5.  Udder health in beef cows and its association with calf growth 
Background
Studies outside the Nordic countries have indicated that subclinical mastitis (measured by milk somatic cell count or the California Mastitis Test), intramammary infections (IMI), or blind quarters in beef cows may have negative effects on beef calf growth. Knowledge on prevalence of such udder health problems in Swedish beef cows is scarce. Therefore, the main aim of this study was to investigate subclinical mastitis, IMI and udder conformation in a number of beef cow herds. Production of β-lactamase in staphylococci was also investigated. Associations between certain cow factors and subclinical mastitis and IMI, and associations between cow and calf factors and 200 day calf weaning weight were also studied. The herds were visited once within a month after calving and once at weaning. Udder examination and quarter milk sampling, for somatic cell count and bacteriology, were performed in 8 to 12 cows per herd and occasion.
Results
Approximately 50%, 40% and 10% of the cows had subclinical mastitis, IMI, and at least one blind quarter, respectively, but the prevalence varied markedly between herds. Intramammary infections (mainly due to staphylococci) were identified in 13-16% of the milk samples. Less than 5% of the staphylococcal isolates produced β-lactamase. Approximately 11% of the cows sampled twice had the same IMI (mostly Staphylococcus aureus) at both samplings. Cow factors of importance for subclinical mastitis and/or IMI were teat and udder shape, breed, parity, presence of blind quarters, and cow hygiene. No significant associations were found between udder health parameters studied and calf weaning weights.
Conclusions
Subclinical mastitis and IMI, but not blind quarters, were common in beef cows, but the prevalence varied markedly between herds. Most IMI were caused by staphylococci and more than 95% of those were sensitive to penicillin. Cows with large funnel-shaped teats or pendulous udder after calving, and cows with blind quarters were at risk of having subclinical mastitis and/or IMI. Poor hygiene was also a risk factor for udder health problems. No significant associations were found between udder health and calf weaning weight. More studies on risk factors are warranted to improve advisory services on awareness and prevention of mastitis in beef cows.
doi:10.1186/1751-0147-56-9
PMCID: PMC3922735  PMID: 24479386
Beef cows; Mastitis; Intra-mammary infections; Blind quarters; Risk factors; Calf weaning weight
6.  Muscle fibre type distribution of the thoracolumbar and hindlimb regions of horses: relating fibre type and functional role 
Background
Although the majority of equine muscles have a mixed fibre type distribution indicative of diverse functional roles, the predominance of a fibre type can indicate the primary function of a muscle. The deep epaxial musculature has an important role in core spinal stability in humans, reflected as a predominantly muscle fibre type (MFT) I or postural fibre type. The fibre type of the deep epaxial musculature has not been determined in horses. The objective of the study was to determine the MFT distribution in selected muscles of thoracolumbar and hindlimb region of horses. This included deep epaxial and hypaxial muscles that were hypothesised to have a postural stabilising role. A second objective was to examine differences in MFT distribution between horses bred for endurance (Arabian) and sprinting (Quarter horse). Muscle biopsy samples were obtained from selected thoracolumbar and hind limb muscles of 5 Quarter horses, 4 Arabians, and 2 Thoroughbreds. The myosin heavy chain distribution was determined by gel electrophoresis. Mann–Whitney rank test was used to compare the proportional MFT and differences between breeds.
Results
Mm. sacrocaudalis dorsalis medialis and diaphragm had the highest proportion of MFT-I. The remaining deep epaxial muscles and the hypaxial muscle m. psoas minor had approximately equal MFT I and II proportions. Mm. psoas major, iliocostalis, longissimus dorsi and the hind limb muscles contained mostly MFT-IIX. The fibre type distribution was similar between Arabians and Quarter horses, although Quarter horses had more MFT-IIX fibres in psoas major (P = 0.02) while Arabians had more MFT-I fibres in m. longissimus dorsi (P = 0.03).
Conclusions
The fibre type distribution of the deep epaxial muscles, mm psoas minor and diaphragm varied from approximately equal MFT-I and II proportions to predominantly MFT-I suggesting a postural stabilising role possibly important in core spinal stability. In contrast the fibre type proportions of mm psoas major, iliocostalis, longissimus dorsi and the hind limb muscles were mainly MFT-II suggesting a locomotory role. Knowledge of fibre type distribution in such a clinically important area can direct diagnosis, prevention and treatment of muscular or neuromotor dysfunction.
doi:10.1186/1751-0147-56-8
PMCID: PMC3922740  PMID: 24468115
Muscle fibre type; Postural; Locomotory; Multifidus; Neuromotor control; Physiotherapy
7.  Salmonella isolated from individual reptiles and environmental samples from terraria in private households in Sweden 
Background
This study investigates Salmonella spp. isolated from privately kept reptiles and from environmental samples such as bedding materials or water from the floor of the enclosures (terraria). It also compares isolation of Salmonella using Modified Semisolid Rappaport-Vassiliadis (MSRV) medium or selective enrichment in Rappaport-Vassiliadis-Soya (RVS) pepton broth. Cloacal swabs or swabs from the cloacal area were collected from 63 individual reptiles belonging to 14 households. All reptiles were from different terraria and from 62 of these, environmental samples were also collected. Sampling were done by the reptile owners according to written instructions and sent by mail immediately after sampling. All but three samples were analyzed within 24 h after collection. Colonies suspected for Salmonella were tested for agglutination and serotyped using the White-Kauffmann-Le Minor scheme. The relative sensitivity (se) and specificity (sp) for MSRV compared with RVS, and the agreement coefficient kappa (κ) were calculated.
Results
Salmonella was isolated from 50/63 (80%) terraria, either from the reptiles (31/63; 49%) or from bedding material (39/62; 63%). The most common subspecies was Salmonella enterica subspecies enterica followed by S. enterica subspecies diarizonae. In reptiles, the most common S. enterica subspecies enterica serovars were Java (n = 4) and Fluntern (n = 4), compared with the serovars Tennessee (n = 10) and Fluntern (n = 10) in the environmental samples. The exact same set of Salmonella subspecies and serovars were not isolated from the individual reptiles and the environmental samples from any of the households. Isolation using MSRV yielded more Salmonella isolates 61/113 (54%) than enrichment in RVS 57/125 (46%). The se was 97.9% (95% Confidence Interval 93.9-100), the sp 78.5% (95% CI 68.5-88.5) and the κ 0.74, indicating substantial agreement between the tests.
Conclusions
Salmonella can be expected to be present in environments where reptiles are kept. This constitutes public health risks and should be considered during handling of the reptiles and during cleaning and disposal of bedding. A combination of different culturing techniques may be used to increase the isolation rate.
doi:10.1186/1751-0147-56-7
PMCID: PMC3922756  PMID: 24461167
Salmonella; Reptile; Public health; Bacteriological culture; Environmental samples; Cloacal samples
8.  Dirofilaria repens infection in a dog imported to Norway 
Dirofilaria repens infection was diagnosed in a dog that had been imported to Norway from Hungary three years previously. The dog was a four-year-old castrated male mixed-breed dog and presented for examination of two masses on the right thoracic wall. Fine needle sampling from the subcutaneous nodules and subsequent cytological examination revealed a high number of microfilariae and a pyogranulomatous inflammation. At re-examination approximately 3 weeks later, both masses had apparently disappeared spontaneously, based on both inspection and palpation. However, examination of peripheral blood by a modified Knott’s test revealed a high number of unsheathed microfilariae with mean length of 360 μm and mean width of 6-7 μm, often with the classic umbrella handle appearance of D. repens. Polymerase chain reaction and sequencing confirmed the D. repens diagnosis. Subcutaneous dirofilariosis caused by D. repens is probably the most common cause of human zoonotic dirofilariosis in Europe, but currently is rarely encountered in northern countries such as Norway. However, travelling, import and relocation of dogs have increased, and thus the geographical range of these parasites is likely to increase from traditionally endemic southern regions. Increasing numbers of autochthonous cases of D. repens infections in dogs have been reported in eastern and central Europe. Although infection with D. repens often induces only mild signs or subclinical infections in dogs, they nevertheless represent a reservoir for zoonotic transmission and thus a public health concern, and, in addition, due to the long prepatent period and the high frequency of subclinical infections or infections with unspecific clinical signs, could easily be missed. Lack of experience and expectation of these parasites may mean that infection is underdiagnosed in veterinary clinics in northern countries. Also, predicted climate changes suggest that conditions in some countries where this infection is currently not endemic are likely to become more suitable for development in the intermediate host, and thus the establishment of the infection in new areas.
doi:10.1186/1751-0147-56-6
PMCID: PMC3933376  PMID: 24447798
Canine; Knott’s test; Molecular methods; Cytology; Dirofilaria repens; Import; Clinical signs; Zoonosis
9.  High feeding intensity increases the severity of fatty liver in the American mink (Neovison vison) with potential ameliorating role for long-chain n-3 polyunsaturated fatty acids 
Background
Rapid body fat mobilization, obesity, and an inadequate supply of n-3 polyunsaturated fatty acids (PUFA) have been suggested to play roles in the etiology of fatty liver in the American mink (Neovison vison). This study examined the effects of feeding intensity and dietary fat source on fatty liver induced by fasting. In a multi-factorial design, 3 different fat sources (herring oil, rich in n-3 PUFA, soya oil, rich in n-6 PUFA, and canola oil, rich in n-9 monounsaturated fatty acids) were fed to mink at a low and high feeding intensity for 10 weeks, followed by an overnight or a 5-day fasting treatment to induce fatty liver.
Results
Fasting led to the development of fatty liver with increased severity in the mink fed at the high feeding intensity. The herring oil diet, high in long-chain n-3 PUFA, was found to decrease the severity of fatty liver in the mink at the high feeding intensity.
Conclusion
Preventing excessive weight gain and increasing dietary intake of n-3 long-chain PUFA may help prevent excessive lipid accumulation during prolonged periods of fasting or inappetence by promoting hepatic fatty acid oxidation.
doi:10.1186/1751-0147-56-5
PMCID: PMC3896742  PMID: 24438337
Fasting; Fatty liver; Mink; Obesity; Polyunsaturated fatty acids
10.  Identification of pregnancy-associated glycoproteins and alpha-fetoprotein in fallow deer (Dama dama) placenta 
Background
This paper describes the isolation and characterization of pregnancy-associated glycoproteins (PAG) from fetal cotyledonary tissue (FCT) and maternal caruncular tissue (MCT) collected from fallow deer (Dama dama) pregnant females. Proteins issued from FCT and MCT were submitted to affinity chromatographies by using Vicia villosa agarose (VVA) or anti-bovine PAG-2 (R#438) coupled to Sepharose 4B gel. Finally, they were characterized by SDS-PAGE and N-terminal microsequencing.
Results
Four distinct fallow deer PAG (fdPAG) sequences were identified and submitted to Swiss-Prot database. Comparison of fdPAG with PAG sequences identified in other ruminant species exhibited 64 to 83% identity. Additionally, alpha-fetoprotein was identified in fetal and maternal tissues.
Conclusion
Our results demonstrate the efficacy of VVA and bovine PAG-2 affinity chromatographies for the isolation of PAG molecules expressed in deer placenta. This is the first report giving four specific amino acid sequences of PAG isolated from feto-maternal junction (FCT and MCT) in the Cervidae family.
doi:10.1186/1751-0147-56-4
PMCID: PMC3896668  PMID: 24410890
Affinity chromatography; Fallow deer; N-terminal microsequencing; Pregnancy-associated glycoprotein; Vicia villosa agarose
11.  Application of optical coherence tomography enhances reproducibility of arthroscopic evaluation of equine joints 
Background
Arthroscopy is widely used in various equine joints for diagnostic and surgical purposes. However, accuracy of defining the extent of cartilage lesions and reproducibility in grading of lesions are not optimal. Therefore, there is a need for new, more quantitative arthroscopic methods. Arthroscopic optical coherence tomography (OCT) imaging is a promising tool introduced for quantitative detection of cartilage degeneration and scoring of the severity of chondral lesions. The aim of this study was to evaluate the inter-investigator agreement and inter-method agreement in grading cartilage lesions by means of conventional arthroscopy and with OCT technique. For this aim, 41 cartilage lesions based on findings in conventional and OCT arthroscopy in 14 equine joints were imaged, blind coded and independently ICRS (International Cartilage Repair Society) scored by three surgeons and one PhD-student.
Results
The intra- and inter-investigator percentages of agreement by means of OCT (68.9% and 43.9%, respectively) were higher than those based on conventional arthroscopic imaging (56.7% and 31.7%, respectively). The intra-investigator Kappa coefficients were 0.709 and 0.565 for OCT and arthroscopy, respectively. Inter-investigator Kappa coefficients were 0.538 and 0.408 for OCT and arthroscopy, respectively.
Conclusions
OCT can enhance reproducibility of arthroscopic evaluation of equine joints.
doi:10.1186/1751-0147-56-3
PMCID: PMC3901375  PMID: 24410869
Arthroscopy; Cartilage lesion; Horse; ICRS-scoring; Optical coherence tomography
12.  Associations between bacterial genotype and outcome of bovine clinical Staphylococcus aureus mastitis 
Background
Staphylococcus aureus is an important cause of clinical mastitis in dairy cows worldwide. The cure rate after antimicrobial treatment of clinical S. aureus mastitis is very variable due to both cow and bacterial factors. Studies have shown that bacterial genotype might affect short-term bacteriological and clinical cure, but the long-term outcome has been less studied. The objectives of this study were to investigate associations between bacterial genotype and long-term outcome of veterinary-treated clinical mastitis (VTCM) caused by S. aureus during a follow-up period of 120 days and to study genotype variation among Swedish S. aureus isolates. S. aureus isolates from cases of VTCM were genotyped by pulsed-field gel electrophoresis. Long-term outcome measurements used were somatic cell count (SCC), additional diagnoses of VTCM, milk yield and culling. Isolates were classified into clusters (>80% similarity) and pulsotypes (100% similarity). Clusters and pulsotypes were grouped according to occurrence. Multivariable mixed-effect linear regression models including cow and bacterial factors with possible influence on SCC or milk yield were used to calculate differences in SCC or milk yield between groups. Additional outcome measures were calculated using a test of proportions.
Results
The isolates (n = 185) were divided into 18 clusters and 29 pulsotypes. Two pulsotypes were classified as common, and were found in 64% of the cases of VTCM. Remaining isolates were classified as less common or rare pulsotypes. The distribution was similar at cluster level. Outcome was calculated from follow-up data on 111 cows. Significantly lower SCC during the follow-up period was found in cows infected with common clusters compared to in cows infected with less common/rare clusters. The proportion of cows with SCC <200 000 cells/ml during the whole follow-up period was significantly higher in the group common clusters than in the group less common/rare clusters. Bacterial genotype did not influence the other outcome parameters.
Conclusions
In Sweden, two S. aureus pulsotypes, identified in about 64% of clinical S. aureus cases, were widespread. Cows infected with the common genotypes had significantly lower SCC during 120 days after treatment compared to cows infected with less common or rare genotypes.
doi:10.1186/1751-0147-56-2
PMCID: PMC3901377  PMID: 24397927
Staphylococcus aureus; Dairy cow; Clinical mastitis; Genotypes; Somatic cell count; Long-term mastitis outcome; Pulsed-Field Gel Electrophoresis; PFGE
13.  The carbon dioxide laser: an alternative surgery technique for the treatment of common cutaneous tumors in dogs 
Background
Tumors of the skin and subcutaneous tissue are the largest group of canine neoplasms. Total excision is still the most effective method for treatment of these skin tumors. For its universal properties the carbon dioxide (CO2) laser appears to be an excellent surgical instrument in veterinary surgery. Laser techniques are alternatives to traditional methods for the surgical management of tumors. The aim of this study was to compare various types of laser techniques in skin oncologic surgery: excision, ablation and mixed technique and to suggest which technique of CO2 laser procedure is the most useful in particular case of tumors in dogs.
Findings
The study was performed on 38 privately-owned dogs with total number of 40 skin tumors of different type removed by various CO2 laser operation techniques from 2010–2013. The treatment effect was based on the surgical wound evaluation, the relative time of healing and possible local recurrence of the tumor after 3 months post surgery. Local recurrence was observed in two cases. The study showed that in 30 cases time needed for complete resection of lesions was less than 10 minutes. Time of healing was longer than 12 days in 6 cases (42.8%) with tumor excision and in 14 cases (87.5%) where excision with ablation technique was performed.
Conclusions
The advantages of the CO2 laser surgery were better hemostasis, precision of working, non-contact dissection, less instruments at the site of operation and minimum traumatization of the surrounding tissues.
doi:10.1186/1751-0147-56-1
PMCID: PMC3896828  PMID: 24393628
CO2 laser surgery; Ablation; Skin tumors; Dogs
14.  Camelus dromedarius brucellosis and its public health associated risks in the Afar National Regional State in northeastern Ethiopia 
Background
A cross-sectional study was carried out in four districts of the Afar region in Ethiopia to determine the prevalence of brucellosis in camels, and to identify risky practices that would facilitate the transmission of zoonoses to humans. This study involved testing 461 camels and interviewing 120 livestock owners. The modified Rose Bengal plate test (mRBPT) and complement fixation test (CFT) were used as screening and confirmatory tests, respectively. SPSS 16 was used to analyze the overall prevalence and potential risk factors for seropositivity, using a multivariable logistic regression analysis.
Results
In the camel herds tested, 5.4% had antibodies against Brucella species, and the district level seroprevalence ranged from 11.7% to 15.5% in camels. The logistic regression model for camels in a herd size > 20 animals (OR = 2.8; 95% CI: 1.16-6.62) and greater than four years of age (OR = 4.9; 95% CI: 1.45-16.82) showed a higher risk of infection when compared to small herds and those ≤ 4 years old. The questionnaire survey revealed that most respondents did not know about the transmission of zoonotic diseases, and that their practices could potentially facilitate the transmission of zoonotic pathogens.
Conclusions
The results of this study revealed that camel brucellosis is prevalent in the study areas. Therefore, there is a need for implementing control measures and increasing public awareness in the prevention methods of brucellosis.
doi:10.1186/1751-0147-55-89
PMCID: PMC3896719  PMID: 24344729
Brucellosis; Camelus dromedarius; CFT; mRBPT; Pastoralists’ risky practices
15.  CPRMethicillin resistant coagulase-negative staphylococci isolated from South Korean ducks exhibiting tremor 
Background
We describe coagulase-negative staphylococci (CoNS) isolates collected from ducklings exhibiting tremor in South Korea over the period of 2010 to 2011. Screening of antimicrobial susceptibility and analysis of SCCmec elements of CoNS were also investigated.
Results
Staphylococcus cohnii was the most frequent staphylococcus (9 isolates) and S. sciuri (4 isolates), S. lentus (3 isolate), S. simulans (1 isolate) and S. epidermidis (1 isolate) were also detected. Among the 15 antimicrobials tested in this study, resistance against oxacillin (15 isolates, 83.3%) was most frequently observed, but only one isolate (SNUDS-1) possessed mecA. This isolate was shown to possess SCCmec type III; the type 3 ccr complex and the class A mec complex.
Conclusions
Based on these results, isolate SNUDS-1 was shown to possess SCCmec type III; the type 3 ccr complex and the class A mec complex. Although the SCCmec type III is not predominant in human, MR-CoNS (Methicillin resistance Coagulase-negative staphylococci) in food animals should be monitored to prevent the dissemination of antimicrobial resistance genes and resistant pathogens to the community.
doi:10.1186/1751-0147-55-88
PMCID: PMC3904464  PMID: 24330456
Methicillin resistance; Coagulase negative staphylococci; Duck; SCCmec complex
16.  Simultaneous subchronic exposure to selenium and diazinon as possible risk factor for osteoporosis in adult male rats 
Background
Osteoporosis and its main health outcome, fragility fractures, are large and escalating health problems. Skeletal damage may be the critical result of low-level prolonged exposure to several xenobiotics in the general population, but the mechanisms of their adverse effects are not clearly understood. The current study was aimed to investigate the possible ability of simultaneous subchronic peroral administration of selenium (Se) and diazinon (DZN) to induce changes in bone of adult male rats.
In our study, twenty 1-month-old male Wistar rats were randomly divided into two experimental groups. In the first group, young males were exposed to 5 mg Na2SeO3/L and 40 mg of DZN/L in drinking water, for 90 days. Ten 1-month-old males without Se and DZN intoxication served as a control group. At the end of the experiment, macroscopic and microscopic structures of the femurs were analysed using analytical scales, sliding instrument, and polarized light microscopy.
Results
The body weight, femoral length and cortical bone thickness were significantly decreased in rats simultaneously exposed to Se and DZN (P < 0.05). These rats also displayed different microstructure in the middle part of the compact bone where vascular canals expanded into central area of substantia compacta. The canals occurred only near endosteal surfaces in rats from the control group. Additionally, a smaller number of primary and secondary osteons, as well as a few resorption lacunae were observed near endosteal surfaces in rats simultaneously administered to Se and DZN. The resorption lacunae as typical structures of bone resorption manifestation are connected with an early stage of osteoporosis. Histomorphometric analysis revealed that area, perimeter, maximum and minimum diameters of primary osteons’ vascular canals were significantly increased (P < 0.05) in the Se-DZN-exposed rats. On the other hand, all measured variables of Haversian canals and secondary osteons were considerable reduced (P < 0.05) in these rats.
Conclusions
Simultaneous subchronic peroral exposure to Se and DZN induces changes in macroscopic and microscopic structures of the femurs in adult male rats, and also it can be considered as possible risk factor for osteoporosis. The current study contributes to the knowledge on damaging impact of several xenobiotics on the bone.
doi:10.1186/1751-0147-55-81
PMCID: PMC3843554  PMID: 24237628
17.  SagE induces highly effective protective immunity against Streptococcus iniae mainly through an immunogenic domain in the extracellular region 
Background
Streptococcus iniae is a Gram-positive bacterium and a severe pathogen of a wide range of farmed fish. S. iniae possesses a virulence-associated streptolysin S cluster composed of several components, one of which is SagE. SagE a transmembrane protein with one major extracellular region named ECR. This study aimed to develop a SagE-based DNA candidate vaccine against streptococcosis and examine the immunoprotective mechanism of the vaccine.
Results
We constructed a DNA vaccine, pSagE, based on the sagE gene and examined its immunological property in a Japanese flounder (Paralichthys olivaceus) model. The results showed that at 7 days post-vaccination, expression of SagE at transcription and translation levels was detected in the tissues of the vaccinated fish. After challenge with S. iniae at one and two months post-vaccination, pSagE-vaccinated fish exhibited relative percent survival (RPS) of 95% and 88% respectively. Immunological analysis showed that (i) pSagE significantly upregulated the expression of a wide range of immune genes, (ii) pSagE induced the production of specific serum antibodies that bound whole-cell S. iniae, and (iii) treatment of S. iniae with pSagE-induced antibodies blocked bacterial invasion of host cells. To localize the immunoprotective domain of SagE, the ECR-expressing DNA vaccine pSagEECR was constructed. Immunization analysis showed that flounder vaccinated with pSagEECR exhibited a RPS of 68%, and that pSagEECR induced serum antibody production and immune gene expression in a manner similar to, though to lower magnitudes than, those induced by pSagE.
Conclusions
We in this study developed a DNA vaccine, pSagE, which induces highly protective immunity against S. iniae. The protective effect of pSagE is probably due to its ability to elicit systemic immune response, in particular that of the humoral branch, which leads to production of specific serum antibodies that impair bacterial infection. These results add insights to the immunoprotective mechanism of fish DNA vaccine.
doi:10.1186/1751-0147-55-78
PMCID: PMC3829104  PMID: 24215645
Streptococcus iniae; DNA vaccine; Immune response; Virulence
18.  Serological markers of Bornavirus infection found in horses in Iceland 
Background
In a stable of eight horses in Northern Iceland, six horses presented with clinical signs, such as ataxia and reduced appetite, leading to euthanasia of one severely affected horse. Serological investigations revealed no evidence of active equine herpes virus type 1 infection, a common source of central nervous system disease in horses, nor equine arteritis virus and West Nile virus. Another neurotropic virus, Borna disease virus, was therefore included in the differential diagnosis list.
Findings
Serological investigations revealed antibodies against Borna disease virus in four of five horses with neurological signs in the affected stable. One horse without clinical signs was seronegative. Four clinically healthy horses in the stable that arrived and were sampled one year after the outbreak were found seronegative, whereas one of four investigated healthy horses in an unaffected stable was seropositive.
Conclusions
This report contains the first evidence of antibodies to Borna disease virus in Iceland. Whether Borna disease virus was the cause of the neurological signs could however not be confirmed by pathology or molecular detection of the virus. As Iceland has very restricted legislation regarding animal imports, the questions of how this virus has entered the country and to what extent markers of Bornavirus infection can be found in humans and animals in Iceland remain to be answered.
doi:10.1186/1751-0147-55-77
PMCID: PMC3828001  PMID: 24180621
Borna disease; Neurological disease; Serology; Epidemiology; Horse
19.  Brain microabscesses in a porcine model of Staphylococcus aureus sepsis 
Background
Sepsis caused by Staphylococcus aureus often leads to brain microabscesses in humans. Animal models of haematogenous brain abscesses would be useful to study this condition in detail. Recently, we developed a model of S. aureus sepsis in pigs and here we report that brain microabscesses develop in pigs with such induced S. aureus sepsis.
Twelve pigs were divided into three groups. Nine pigs received an intravenous inoculation of S. aureus once at time 0 h (group 1) or twice at time 0 h and 12 h (groups 2 and 3). In each group the fourth pig served as control. The pigs were euthanized at time 12 h (Group 1), 24 h (Group 2) and 48 h (Group 3) after the first inoculation. The brains were collected and examined histopathologically.
Results
All inoculated pigs developed sepsis and seven out of nine pigs developed brain microabscesses. The microabscesses contained S. aureus and were located in the prosencephalon and mesencephalon. Chorioditis and meningitis occurred from 12 h after inoculation.
Conclusions
Pigs with experimental S. aureus sepsis often develop brain microabscesses. The porcine brain pathology mirrors the findings in human sepsis patients. We therefore suggest the pig as a useful animal model of the development of brain microabscesses caused by S. aureus sepsis.
doi:10.1186/1751-0147-55-76
PMCID: PMC3843533  PMID: 24176029
Pig; Staphylococcus aureus; Brain; Model; Sepsis; Abscess; Haematogenous
20.  The use of the rapid osmotic fragility test as an additional test to diagnose canine immune-mediated haemolytic anaemia 
Background
Diagnosing canine immune-mediated haemolytic anaemia (IMHA) is often challenging because all currently available tests have their limitations. Dogs with IMHA often have an increased erythrocyte osmotic fragility (OF), a characteristic that is sometimes used in the diagnosis of IMHA. Since the classic osmotic fragility test (COFT) is time-consuming and requires specialized equipment, an easy and less labour-intensive rapid osmotic fragility test (ROFT) has been used in some countries, but its diagnostic value has not yet been investigated.
This study aimed to evaluate erythrocyte osmotic fragility in dogs with and without IMHA, to compare results of the classic (COFT) and rapid (ROFT) test and to assess the value of the ROFT as diagnostic test for canine IMHA.
Nineteen dogs with IMHA (group 1a), 21 anaemic dogs without IMHA (group 1b), 8 dogs with microcytosis (group 2), 13 hyperlipemic dogs (group 3), 10 dogs with lymphoma (group 4), 8 dogs with an infection (group 5) and 13 healthy dogs (group 6) were included.
In all dogs, blood smear examination, in-saline auto-agglutination test, Coombs’ test, COFT and ROFT were performed. In the COFT, OF5, OF50 and OF90 were defined as the NaCl concentrations at which respectively 5, 50 and 90% of erythrocytes were haemolysed.
Results
Compared with healthy dogs, OF5 and OF50 were significantly higher in group 1a (P < 0.001) and OF5 was significantly higher in group 3 (P = 0.0266). The ROFT was positive in 17 dogs with IMHA, 10 hyperlipemic dogs, one anaemic dog without IMHA and one healthy dog.
Conclusions
Osmotic fragility was increased in the majority of dogs with IMHA and in dogs with hyperlipidemia, but not in dogs with microcytosis, lymphoma or an infection. Although more detailed information was obtained about the osmotic fragility by using the COFT, the COFT and ROFT gave similar results. The ROFT does not require specialized equipment, is rapid and easy to perform and can be used easily in daily practice. Although, the ROFT cannot replace other diagnostic tests, it may be a valuable additional tool to diagnose canine IMHA.
doi:10.1186/1751-0147-55-74
PMCID: PMC3816578  PMID: 24160183
Osmotic fragility; Canine immune-mediated haemolytic anaemia; Hyperlipidemia
21.  Evaluation of farmers’ diagnostic performance for detection of diarrhoea in nursery pigs using digital pictures of faecal pools 
Background
Overconsumption of antibiotics in the pig industry is of concern in relation to antimicrobial resistance. False positive disease diagnosis may result in the treatment of healthy animals. In Denmark, diarrhoea is the most common cause of antibiotic treatment in pigs. Farm personnel are not professional clinicians, which could result in inappropriate antibiotic treatments of diarrhoea.
The primary objectives of this pilot study using digital pictures of faecal pools was to evaluate farmers’ diagnostic performance in the assessment of faecal consistency in nursery pigs and to investigate the effect of different co-variables, including practical experience. A secondary objective was to compare the diagnostic performance of farmers with that of veterinarians.
At a pig congress, observers (farm personnel and veterinarians) working professionally with pigs participated in a faecal consistency test consisting of 16 pictures of faecal pools (eight diarrhoeic and eight non-diarrhoeic). The faecal pools had previously been collected and subjected to faecal dry matter determination. The true status of the faecal pools was determined by the faecal dry matter content (diarrhoea: faecal dry matter ≤ 18%). The true status was used to evaluate the farmers’ and veterinarians’ diagnostic performance.
Results
A total of 119 farmers and 18 veterinarians were included in the statistical analysis. For the farmers, the mean proportion of faecal pools assessed as diarrhoeic was 0.48, the mean proportion of correctly classified faecal pools was 0.84, the mean diagnostic sensitivity was 0.83 and the mean diagnostic specificity was 0.86. Farmers with less than four years of practical experience detected clinical diarrhoea more accurately than farmers with more than four years of practical experience (p < 0.05). No significantly differences between farmers and veterinarians was observed (p > 0.20).
Conclusions
The results, using digital pictures of faecal pools, suggest that farmers and veterinarians have similar diagnostic performance in relation to diarrhoea. False positive classification of non-diarrhoeic pigs appears to be a larger problem than false negative classification of diarrhoeic pigs under Danish conditions. If these results can be confirmed under practical conditions, training in, and validation of, clinical diagnoses may be an important factor in reducing antibiotic consumption in the pig industry.
doi:10.1186/1751-0147-55-72
PMCID: PMC3819656  PMID: 24138810
Clinical signs; Diagnostic performance; Farmers; Faecal consistency; Diarrhoea; Pigs
22.  Looking for prognosticators in ovine anaplasmosis: discriminant analysis of clinical and haematological parameters in lambs belonging to differently susceptible breeds experimentally infected with Anaplasma ovis 
Background
A study was carried out to evaluate the response of different native sheep breeds to experimental infection with Anaplasma ovis, the most prevalent sheep tick-borne pathogen in Apulia (Southern Italy). Thirty-four lambs belonging to a Northern European breed (Suffolk) and two Southern Italian breeds (Comisana and Altamurana) were infected. Eleven clinical as well as haematological parameters were monitored at different temporal resolutions on the same subjects before and after the infection, resulting in a data set of 435 observations. The present work, aiming to further the research, presents the results of a multivariate analysis carried out to identify which parameters out of the eleven considered are the most reliable parameters to be considered as markers of the disease phenotype as well as prognosticators of practical clinical importance.
Results
Data were analysed by discriminant analysis. Out of the eleven considered variables (red blood cells, packed cell volume, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin content, haemoglobin concentration, white blood cells, neutrophils, leukocytes, platelets, rectal temperature), only seven were included in the step-wise model since significantly increasing the Mahlanobis distance between the two closest groups. Both discriminant functions resulted to be highly significant (P < 0.0001) and the percentage of variation accounted for by the first discriminant function was 63.6% of the variance in the grouping variable.
Conclusions
Taken together, the observed results stress the marked differentiation among the three breeds in terms of physio-pathological phenotypes indicating packed cell volume and red blood cell count as the most informative parameters in the routine clinical practice for A. ovis infection in sheep.
doi:10.1186/1751-0147-55-71
PMCID: PMC3850957  PMID: 24053615
Anaplasma ovis; Tolerance to anaplasmosis; Native sheep breeds; Clinical prognosticators
23.  The first detection of influenza in the Finnish pig population: a retrospective study 
Background
Swine influenza is an infectious acute respiratory disease of pigs caused by influenza A virus. We investigated the time of entry of swine influenza into the Finnish pig population. We also describe the molecular detection of two types of influenza A (H1N1) viruses in porcine samples submitted in 2009 and 2010.
This retrospective study was based on three categories of samples: blood samples collected for disease monitoring from pigs at major slaughterhouses from 2007 to 2009; blood samples from pigs in farms with a special health status taken in 2008 and 2009; and diagnostic blood samples from pigs in farms with clinical signs of respiratory disease in 2008 and 2009.
The blood samples were tested for influenza A antibodies with an antibody ELISA. Positive samples were further analyzed for H1N1, H3N2, and H1N2 antibodies with a hemagglutination inhibition test.
Diagnostic samples for virus detection were subjected to influenza A M-gene-specific real-time RT-PCR and to pandemic influenza A H1N1-specific real-time RT-PCR. Positive samples were further analyzed with RT-PCRs designed for this purpose, and the PCR products were sequenced and sequences analyzed phylogenetically.
Results
In the blood samples from pigs in special health class farms producing replacement animals and in diagnostic blood samples, the first serologically positive samples originated from the period July–August 2008. In samples collected for disease monitoring, < 0.1%, 0% and 16% were positive for antibodies against influenza A H1N1 in the HI test in 2007, 2008, and 2009, respectively.
Swine influenza A virus of avian-like H1N1 was first detected in diagnostic samples in February 2009. In 2009 and 2010, the avian-like H1N1 virus was detected on 12 and two farms, respectively. The pandemic H1N1 virus (A(H1N1)pdm09) was detected on one pig farm in 2009 and on two farms in 2010.
Conclusions
Based on our study, swine influenza of avian-like H1N1 virus was introduced into the Finnish pig population in 2008 and A(H1N1)pdm09 virus in 2009. The source of avian-like H1N1 infection could not be determined. Cases of pandemic H1N1 in pigs coincided with the period when the A(H1N1)pdm09 virus was spread in humans in Finland.
doi:10.1186/1751-0147-55-69
PMCID: PMC3850993  PMID: 24047612
Porcine; Influenza A; H1N1; Serology; Phylogenetic analysis; Finland
24.  Ultrasonography of the spleen, liver, gallbladder, caudal vena cava and portal vein in healthy calves from birth to 104 days of age 
Background
Many of the ultrasonographic abdominal findings of adult cattle probably also apply to calves. However, significant changes associated with ruminal growth and transition from a milk to a roughage diet occur in young calves during the first few months, and it can be expected that ultrasonographic features of organs adjacent to the rumen such as spleen and liver also undergo significant changes. These have not been investigated to date and therefore the goal of this study was to describe ultrasonographic findings of the spleen, liver, gallbladder, caudal vena cava and portal vein in six healthy calves from birth to 104 days of age. Standing calves were examined ultrasonographically six times at three-week intervals starting on the first or second day of life using a 5.0-MHz transducer and techniques described previously.
Results
The spleen was imaged on the left at the 5th to 12th intercostal spaces. The dorsal and ventral visible limits ran from cranioventral to caudodorsal because of superimposition of the lungs. The size of the spleen was largest at the 7th and 8th intercostal spaces and the maximum thickness was measured at the 9th to 12th intercostal spaces. The liver was seen in all calves on the right and could be imaged at the 5th to 12th intercostal spaces and the area caudal to the last rib. Similar to the spleen, the dorsal visible margin of the liver ran parallel to the ventral border of the lungs. The visible size of the liver was largest at the 8th to 11th intercostal spaces and the maximum thickness was measured at the 8th and 9th intercostal spaces. The parenchymal pattern consisted of numerous fine echoes homogeneously distributed over the entire organ. The gallbladder was most commonly seen at the 9th intercostal space and was circular, oval or pear-shaped on ultrasonograms. It extended beyond the ventral border of the liver depending on the amount of bile. The caudal vena cava was triangular in cross section but sometimes had a round or oval profile and was always seen in at least one intercostal space. The maximum circumference was measured at the 10th and 11th intercostal spaces. The portal vein was circular or oval in cross section and was characterised by stellate ramifications branching into the liver parenchyma. The portal vein could always be imaged at the 7th to 11th intercostal spaces and its mean diameter at the 9th to 11th intercostal spaces ranged from 1.2 cm to 1.8 cm.
Conclusions
The ultrasonographic findings of the spleen, liver, gallbladder, caudal vena cava and portal vein in six healthy Holstein-Friesian calves from birth to 104 days of age serve as reference values for the examination of these anatomical structures in diseased calves.
doi:10.1186/1751-0147-55-68
PMCID: PMC3852301  PMID: 24040969
25.  Haemorrhagic pneumonia in sled dogs caused by Streptococcus equi subsp. zooepidemicus - one fatality and two full recoveries: a case report 
In spite of yearly vaccination, outbreaks of canine infectious respiratory disease are periodically seen amongst domestic dogs. These infections compromise host defense mechanisms, and, when combined with other stressful events, allow opportunistic pathogens like Streptococcus equi subsp. zooepidemicus to create serious disease. Early recognition and treatment are tremendously important for a successful outcome in these cases. A polyvalent vaccine was given to 22 racing dogs three days after a competition, followed by two days of rest, and then the dogs were returned to regular training. Coughing was noticed among the dogs four days after immunisation. Three days after this outbreak one of the dogs was unusually silent and was found dead the next morning. Simultaneously two other dogs developed haemorrhagic expectorate, depression and dyspnea and were brought in to the veterinary hospital. Streptococcus equi subsp. zooepidemicus was isolated in pure culture from all three cases. They were treated and rehabilitated successfully, and won a sledge race three months later. This paper discusses the necropsy results, treatment regime, rehabilitation and the chronology of vaccination, stressful events and disease.
doi:10.1186/1751-0147-55-67
PMCID: PMC3852515  PMID: 24020788
Respiratory infection; Canine; Rehabilitation; Treatment; Immunology; Histology

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