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1.  Is the presence of abnormal prion protein in the renal glomeruli of feline species presenting with FSE authentic? 
In a recent paper written by Hilbe et al (BMC vet res, 2009), the nature and specificity of the prion protein deposition in the kidney of feline species affected with feline spongiform encephalopathy (FSE) were clearly considered doubtful. This article was brought to our attention because we published several years ago an immunodetection of abnormal prion protein in the kidney of a cheetah affected with FSE. At this time we were convinced of its specificity but without having all the possibilities to demonstrate it. As previously published by another group, the presence of abnormal prion protein in some renal glomeruli in domestic cats affected with FSE is indeed generally considered as doubtful mainly because of low intensity detected in this organ and because control kidneys from safe animals present also a weak prion immunolabelling. Here we come back on these studies and thought it would be helpful to relay our last data to the readers of BMC Vet res for future reference on this subject.
Here we come back on our material as it is possible to study and demonstrate the specificity of prion immunodetection using the PET-Blot method (Paraffin Embedded Tissue - Blot). It is admitted that this method allows detecting the Proteinase K (PK) resistant form of the abnormal prion protein (PrPres) without any confusion with unspecific immunoreaction. We re-analysed the kidney tissue versus adrenal gland and brain samples from the same cheetah affected with TSE using this PET-Blot method. The PET-Blot analysis revealed specific PrPres detection within the brain, adrenal gland and some glomeruli of the kidney, with a complete identicalness compared to our previous detection using immunohistochemistry. In conclusion, these new data enable us to confirm with assurance the presence of specific abnormal prion protein in the adrenal gland and in the kidney of the cheetah affected with FSE. It also emphasizes the usefulness for the re-examination of any available tissue blocks with the PET-Blot method as a sensitive complementary tool in case of doubtful PrP IHC results.
PMCID: PMC2923130  PMID: 20684771
3.  Transmission of border disease virus from a persistently infected calf to seronegative heifers in early pregnancy 
This study describes the transmission of border disease virus (BDV) from a persistently infected calf to seronegative heifers in early pregnancy, resulting in persistently infected fetuses. On day 50 of pregnancy (= day 0 of the infection phase), six heifers were co-housed in a free stall with a bull calf persistently infected with BDV (pi BVD) for 60 days. The heifers underwent daily clinical examination, and blood samples were collected regularly for detection of pestiviral RNA and anti-pestivirus antibodies. After day 60 (= day 110 of pregnancy), the heifers were slaughtered, and the fetuses and placentae underwent post-mortem and immunohistochemical examination and RT-PCR for viral RNA detection.
Three heifers had mild viraemia from day 8 to day 14, and by day 40 all heifers had pestivirus antibodies identified as anti-BDV antibodies in the serum neutralisation test. The placenta of the three viraemic heifers had histological evidence of inflammation, and fetal organs from these heifers were positive for pestivirus antigen by immunohistochemical examination and for BD viral RNA by RT-PCR and sequencing. Thus, co-housing of heifers in early pregnancy with a pi-BDV calf led to seroconversion in all heifers and persistent fetal infection in three.
Considering that pi-BDV cattle can infect other cattle and lead to persistent infection of the fetus in pregnant cows, BDV should not be ignored in the context of the mandatory BVDV eradication and monitoring program. This strongly suggests that BDV should be taken into account in BVD eradication and control programs.
PMCID: PMC4336514
Cattle; Border disease; Early pregnancy; Persistent infection; BVDV; Pestivirus
4.  BMC Veterinary Research reviewer acknowledgement 2014 
PMCID: PMC4332966
5.  Establishment of a mouse model to express bovine CD14 short hairpin RNA 
Cluster of differentiation 14 (CD14) functions as a co-receptor for Toll-like receptor (TLR)-4 and myeloid differentiation factor (MD)-2 in detecting bacterial lipopolysaccharide. Together, these complexes promote the phagocytosis and digestion of Gram-negative bacteria, and initiate immune responses. To date, much of our understanding of CD14 function during Gram-negative bacterial inflammation comes from studies on mouse knockout models and cell transfection. To identify the effect of CD14 knockdown in this process in large livestock animals, we established a mouse model expressing bovine CD14 short hairpin (sh) RNA. shRNA fragments targeting bovine CD14 were screened by co-transfection in HEK 293 cells, and the most effective CD14 shRNA fragment was cloned into the eukaryotic expression vector pSilencer4.1-CD14 shRNA-IRES (internal ribosome entry site) and transferred into mouse zygotes by pronuclear microinjection to obtain transgenic mice. Expression of the enhanced green fluorescent protein (EGFP) reporter and genes related to the TLR4 signaling pathway was detected by immunohistochemistry (IHC) and quantitative polymerase chain reaction (PCR), respectively.
One effective shRNA fragment (shRNA-674) targeting bovine CD14 was obtained, the sequence of which was shown to be conserved between cows, buffalos, sheep, and humans. Thirty-seven founder pups were obtained by pronuclear microinjection, of which three were positive for the transgene. In the F1 generation, 11 of 33 mice (33%) were positive for the transgene as detected by PCR. IHC analysis detected exogenous EGFP expression in the liver, kidney, and spleen of transgenic F1 mice, indicating that they were chimeric. The expression of endogenous CD14 mRNA in the heart, liver, spleen, lung, and kidney of transgenic F1 mice was decreased 8-, 3-, 19.5-, 6-, and 11-fold, respectively. The expression patterns of endogenous MD-2, TLR4, interleukin-6 and tumor necrosis factor-α genes in transgenic mice also varied.
This study confirms that transgenic mice expressing bovine CD14 shRNA can be generated by pronuclear microinjection, and demonstrates inhibited endogenous mouse CD14 expression that alters gene expression related to the TLR4 signaling pathway.
PMCID: PMC4332730
Bovine CD14; shRNA; Mouse model; TLR4; Gene expression
6.  Investigating the introduction of porcine epidemic diarrhea virus into an Ohio swine operation 
Porcine Epidemic Diarrhea virus (PEDV) is a highly transmissible coronavirus that causes a severe enteric disease that is particularly deadly for neonatal piglets. Since its introduction to the United States in 2013, PEDV has spread quickly across the country and has caused significant financial losses to pork producers. With no fully licensed vaccines currently available in the United States, prevention and control of PEDV disease is heavily reliant on biosecurity measures. Despite proven, effective biosecurity practices, multiple sites and production stages, within and across designated production flows in an Ohio swine operation broke with confirmed PEDV in January 2014, leading the producer and attending veterinarian to investigate the route of introduction.
Case presentation
On January 12, 2014, several sows within a production flow were noted with signs of enteric illness. Within a few days, illness had spread to most of the sows in the facility and was confirmed by RT-PCR to be PEDV. Within a short time period, confirmed disease was present on multiple sites within and across breeding and post weaning production flows of the operation and mortality approached 100% in neonatal piglets. After an epidemiologic investigation, an outsourced, pelleted piglet diet was identified for assessment, and a bioassay, where naïve piglets were fed the suspected feed pellets, was initiated to test the pellets for infectious PEDV.
The epidemiological investigation provided strong evidence for contaminated feed as the source of the outbreak. In addition, feed pellets collected from unopened bags at the affected sites tested positive for PEDV using RT-PCR. However, the bioassay study was not able to show infectivity when feeding the suspected feed pellets to a small number of naïve piglets. The results highlight the critical need for surveillance of feed and feed components to further define transmission avenues in an effort to limit the spread of PEDV throughout the U.S. swine industry.
PMCID: PMC4334577
Feed; PEDV; Swine
7.  Suitability of sentinel abattoirs for syndromic surveillance using provincially inspected bovine abattoir condemnation data 
Sentinel surveillance has previously been used to monitor and identify disease outbreaks in both human and animal contexts. Three approaches for the selection of sentinel sites are proposed and evaluated regarding their ability to capture overall respiratory disease trends using provincial abattoir condemnation data from all abattoirs open throughout the study for use in a sentinel syndromic surveillance system.
All three sentinel selection criteria approaches resulted in the identification of sentinel abattoirs that captured overall temporal trends in condemnation rates similar to those reported by the full set of abattoirs. However, all selection approaches tended to overestimate the condemnation rates of the full dataset by 1.4 to as high as 3.8 times for cows, heifers and steers. Given the results, the selection approach using abattoirs open all weeks had the closest approximation of temporal trends when compared to the full set of abattoirs.
Sentinel abattoirs show promise for integration into a food animal syndromic surveillance system using Ontario provincial abattoir condemnation data. While all selection approaches tended to overestimate the condemnation rates of the full dataset to some degree, the abattoirs open all weeks selection approach appeared to best capture the overall seasonal and temporal trends of the full dataset and would be the most suitable approach for sentinel abattoir selection.
PMCID: PMC4336523
8.  Development of intestinal microflora and occurrence of diarrhoea in sucking foals: effects of Bacillus cereus var. toyoi supplementation 
Almost all foals develop transient diarrhoea within the first weeks of life. Studies indicated different viral, bacterial, and parasitic causes, such as rotavirus, Clostridium perfringens, Escherichia coli, and Cryptosporidium are discussed. But little is known about the development of intestinal microflora in foals. The present study investigated whether the supplementation with Bacillus cereus var. toyoi would modify the developing intestinal microflora and consequently reduce diarrhoea in foals. From birth, the foals were randomly assigned to three treatment groups: placebo (10 mL isotonic NaCl, n = 8), low dosage (LD; 5 × 108 cfu B. cereus var. toyoi, n = 7) and high dosage (HD; 2 × 109 cfu B. cereus var. toyoi, n = 10). Treatment groups were supplemented orally once a day for 58 days. Faeces scoring and sampling were performed within the first 24 h after birth and on day 9, 16, 23, 30, 44, 58 of the foal’s life and also on the first day of diarrhoea. Culture-plate methods were used to analyse the bacterial microflora.
Eighty-eight per cent of the foals developed diarrhoea (placebo 7/8, LD 5/7, HD 10/10) during the first 58 days of life. Bacillus cereus var. toyoi supplementation had no effect on bacterial microflora. Clostridium perfringens and enterobacteria were equally prevalent in foals with diarrhoea and those who were not afflicted.
We conclude that the supplementation of B. cereus var. toyoi had no effect on the occurrence of diarrhoea and health status in the foals.
Electronic supplementary material
The online version of this article (doi:10.1186/s12917-015-0355-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4333172
Microflora; Foal; Diarrhoea; Probiotic; Bacillus cereus var. toyoi
9.  Natural Besnoitia besnoiti infections in cattle: hematological alterations and changes in serum chemistry and enzyme activities 
The emerging disease bovine besnoitiosis is caused by the apicomplexan parasite Besnoitia besnoiti. Clinical signs of acute besnoitiosis are pyrexia, anorexia and subcutaneous edema. In subacute and chronic besnoitiosis parasitic cysts arise in a variety of tissues and affected cattle display skin lesions and weight loss. In all stages of bovine besnoitiosis, lesions can be found in many organ systems and therefore presumably alter a variety of laboratory parameters. In this study, the impact of naturally acquired acute, subacute and chronic bovine besnoitiosis on hematologic parameters, serum chemistry, and enzyme activities was investigated. Laboratory parameters of two Simmental heifers and two Limousin cows were monitored during acute, subacute and chronic besnoitiosis and in another Simmental heifer during subclinical besnoitiosis. To determine aberrations of laboratory parameters, values were compared with reference ranges obtained from B. besnoiti negative Simmentals (224 samples of nine animals) and Limousins (41 animals). Further, laboratory parameters of B. besnoiti seropositive Limousin cows (54 animals; 32 of these showing clinical signs) and healthy B. besnoiti seronegative Limousin cows (41 animals) were compared.
During acute and subacute besnoitiosis, a reduction of leukocyte and erythrocyte concentrations, hematocrit, serum albumin, urea, magnesium, and calcium concentrations were observed. Serum total protein, globulin, total bilirubin and creatinine concentrations were increased and aspartate transaminase (AST) and creatine kinase (CK) activities were elevated. In chronic besnoitiosis, erythrocyte parameters were statistically significantly lower, and total protein and globulin concentrations were significantly higher in B. besnoiti seropositive compared with B. besnoiti seronegative Limousin cows.
In this study, altered laboratory parameters during the course of naturally acquired acute, subacute and chronic bovine besnoitiosis are described for the first time. Only a few animals were examined in acute and subacute besnoitiosis, however the alterations of laboratory parameters during these stages reflected i) the acute inflammatory state (e.g. high levels of serum globulin fractions), ii) clinical findings such as disturbed condition (e.g. bilirubin concentrations), and iii) lesions such as muscle necroses described in the literature (e.g. AST or CK activities). Chronic besnoitiosis led to typical alterations of chronic inflammatory diseases like hyper-(gamma)-globulinemia or reduced erythrocyte concentrations.
PMCID: PMC4336505
Bovine besnoitiosis; Besnoitia besnoiti; Hematological and biochemical parameters; Cattle
10.  Seroepidemiology of leptospirosis in dogs from rural and slum communities of Los Rios Region, Chile 
Leptospirosis is a zoonotic disease of global importance and often neglected as a public health problem due to lack of awareness, under-diagnosis and under-reporting. Animals serve as a source of transmission through the shedding of Leptospira in their urine. Because of their proximity to humans, dogs may play a role in human infections. In order to assess and mitigate leptospirosis in dogs and the risk of transmission to humans it is important to understand the epidemiology of leptospirosis under natural conditions. This study aimed to characterize leptospirosis in owned dogs from three distinct community types. Blood, dog and household data were collected from 265 dogs in 190 households from 12 communities representing farms, rural villages, and urban slums in the Los Rios region, Chile. Serologic profiles with a 20-serovar microagglutination test panel were obtained. Binomial and multinomial logistic regression models were used to evaluate the associations between spatial, ecological, socio-economic variables and overall seropositivity as well as seropositivity to serogroup Canicola.
Results from 247 dogs with no history of vaccination were used. Overall seroprevalence was 25.1% (62/247) with significant differences by community type: 10.9% (9/82) in dogs from farms, 22.3% (21/94) from rural villages, and 45.1% (32/71) from urban slums (p <0.001). This trend by community type was also observed for dogs with evidence of seropositivity to the Canicola serogroup. Factors associated with seropositive dogs included dog density and precipitation two-weeks prior to sampling. Presence of Leptospira positive puddles collected from the peri-domestic household environment was also associated with increased seropositivity.
Results suggest that leptospirosis is actively maintained in the dog population in this study region with notably distinct patterns by community type. Dog populations from rural villages, and urban slums in particular, showed evidence of high levels of transmission probably as a result of the combined effects of dog living conditions as well as community-level ecological and environmental factors.
Electronic supplementary material
The online version of this article (doi:10.1186/s12917-015-0341-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4329218
Canine leptospirosis; Leptospira; Canicola serogroup; Zoonosis; Ecology epidemiology; Chile
11.  Comparison of risk factors for seropositivity to feline immunodeficiency virus and feline leukemia virus among cats: a case-case study 
Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are reported to have similar risk factors and similar recommendations apply to manage infected cats. However, some contrasting evidence exists in the literature with regard to commonly reported risk factors. In this study, we investigated whether the known risk factors for FIV and FeLV infections have a stronger effect for either infection. This retrospective study included samples from 696 cats seropositive for FIV and 593 cats seropositive for FeLV from the United States and Canada. Data were collected during two cross sectional studies, where cats were tested using IDEXX FIV/FeLV ELISA kits. To compare the effect of known risk factors for FIV infection compared to FeLV, using a case-case study design, random intercept logistic regression models were fit including cats’ age, sex, neuter status, outdoor exposure, health status and type of testing facility as independent variables. A random intercept for testing facility was included to account for clustering expected in testing practices at the individual clinics and shelters.
In the multivariable random intercept model, the odds of FIV compared to FeLV positive ELISA results were greater for adults (OR = 2.09, CI: 1.50-2.92), intact males (OR = 3.14, CI: 1.85-3.76), neutered males (OR = 2.68, CI: 1.44- 3.14), cats with outdoor access (OR = 2.58, CI: 1.85-3.76) and lower for cats with clinical illness (OR = 0.60, 95% CI: 0.52-0.90). The variance components obtained from the model indicated clustering at the testing facility level.
Risk factors that have a greater effect on FIV seropositivity include adulthood, being male (neutered or not) and having access to outdoors, while clinical illness was a stronger predictor for FeLV seropositivity. Further studies are warranted to assess the implications of these results for the management and control of these infections.
PMCID: PMC4332748
Cat; Epidemiology; Retrovirus; FIV; FeLV
12.  Efficacy of multivalent, modified- live virus (MLV) vaccines administered to early weaned beef calves subsequently challenged with virulent Bovine viral diarrhea virus type 2 
Vaccination of young calves against Bovine viral diarrhea virus (BVDV) is desirable in dairy and beef operations to reduce clinical disease and prevent spread of the virus among cattle. Although protection from clinical disease by multivalent, modified-live virus (MLV) vaccines has been demonstrated, the ability of MLV vaccines to prevent viremia and viral shedding in young calves possessing passive immunity is not known. The purpose of this study was to compare the ability of three different MLV vaccines to prevent clinical disease, viremia, and virus shedding in early weaned beef calves possessing maternal immunity that were vaccinated once at 45 days prior to challenge with virulent BVDV 2.
At 45 days following vaccination, calves that received vaccines B and C had significantly higher BVDV 1 and BVDV 2 serum antibody titers compared with control calves. Serum antibody titers for BVDV 1 and BVDV 2 were not significantly different between control calves and calves that received vaccine D. Following BVDV 2 challenge, a higher proportion of control calves and calves that received vaccine D presented viremia and shed virus compared with calves that received vaccines B and C. Rectal temperatures and clinical scores were not significantly different between groups at any time period. Calves that received vaccines B and C had significantly higher mean body weights at BVDV 2 challenge and at the end of the study compared with control calves.
Moderate to low maternally-derived BVDV antibody levels protected all calves against severe clinical disease after challenge with virulent BVDV 2. Vaccines B and C induced a greater antibody response to BVDV 1 and BVDV 2, and resulted in reduced viremia and virus shedding in vaccinated calves after challenge indicating a greater efficacy in preventing virus transmission and reducing negative effects of viremia.
PMCID: PMC4334402
Early weaning; BVDV; MLV; Vaccine; Antibody titres; Virus; Isolation; Shedding
13.  A comparison of pre and post-operative vedaprofen with ketoprofen for pain control in dogs 
This prospective randomized blinded clinical study aimed to investigate the potential of vedaprofen for preventive analgesia, comparing its analgesic effects with ketoprofen administered post-operatively in dogs undergoing maxillectomy or mandibulectomy.
Pain control was effective and rescue analgesia was not necessary in any group. Pain scores were not significantly different between groups. The respiratory rate and rectal temperature were decreased in all groups at extubation until 6 hours post-extubation compared to baseline. Cortisol and epinephrine levels were increased only at 0.5 hours after extubation in all groups compared to baseline.
Vedaprofen did not present any preventive analgesic effect. Pre- and postoperative vedaprofen were as effective as ketoprofen for postoperative pain control.
Electronic supplementary material
The online version of this article (doi:10.1186/s12917-015-0338-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4324799
Non-steroidal anti-inflammatory drug; Sublingual; Analgesia; Preventive; Preemptive
14.  The effect of Zhangfei/CREBZF on cell growth, differentiation, apoptosis, migration, and the unfolded protein response in several canine osteosarcoma cell lines 
We had previously shown that the bLZip domain-containing transcription factor, Zhangfei/CREBZF inhibits the growth and the unfolded protein response (UPR) in cells of the D–17 canine osteosarcoma (OS) line and that the effects of Zhangfei are mediated by it stabilizing the tumour suppressor protein p53. To determine if our observations with D-17 cells applied more universally to canine OS, we examined three other independently isolated canine OS cell lines—Abrams, McKinley and Gracie.
Like D–17, the three cell lines expressed p53 proteins that were capable of activating promoters with p53 response elements on their own, and synergistically with Zhangfei. Furthermore, as with D–17 cells, Zhangfei suppressed the growth and UPR-related transcripts in the OS cell lines. Zhangfei also induced the activation of osteocalcin expression, a marker of osteoblast differentiation and triggered programmed cell death.
Osteosarcomas are common malignancies in large breeds of dogs. Although there has been dramatic progress in their treatment, these therapies often fail, leading to recurrence of the tumour and metastatic spread. Our results indicate that induction of the expression of Zhangfei in OS, where p53 is functional, may be an effective modality for the treatment of OS.
PMCID: PMC4326286
Canine osteosarcoma; Zhangfei/CREBZF; p53; Apoptosis; Osteocalcin
15.  Development of vaccine-induced immunity against TRT in turkeys depends remarkably on the level of maternal antibodies and the age of birds on the day of vaccination 
Avian Metapneumovirus (aMPV) infections are a huge economical issue for the poultry industry worldwide. Although maternal antibodies do not protect turkey poults against turkey rhinotracheitis (TRT), almost no studies have been conducted so far regarding the impact of these antibodies on vaccine induced immunity development against aMPV infection. We conducted four experiments on commercial turkeys aimed at comparing local humoral and cell mediated immune response of maternally delivered anti-aMPV antibody positive (MDA+; Experiment I and II) and negative (MDA-; Experiment III and IV) turkeys following vaccination with an attenuated live aMPV subtype A vaccine at the day of hatch (Experiment I and III) or at two weeks of age (Experiment II and IV).
Regardless of the birds’ age, vaccination of MDA- turkeys resulted in strong stimulation of CD8+ T lymphocytes in the Harderian gland and tracheal mucosa, whereas vaccination of MDA+ birds stimulated mainly CD4+ T cells in those structures. An increase in the level of anti-aMPV IgY antibodies was noted in the serum (but not in tracheal washings) as early as 7 days after vaccination, but only in birds possessing low levels (MDA+ birds vaccinated at 2 weeks of age) or no maternal anti-aMPV antibodies at the time of vaccination. In MDA+ turkeys vaccinated at hatch, the decrease in serum levels of maternal anti-aMPV antibodies proceeded faster (in comparison to control group), which, together with faster viral clearance, indicates that maternal antibodies can inhibit vaccine virus replication and influence the development of vaccine-induced immunity.
This study provides the first documented evidence that the frequency of TRT outbreaks in the field and/or failure of TRT vaccination could be correlated with differences in the immunological status and/or age of vaccinated turkeys.
PMCID: PMC4326515
Avian metapneumovirus; Turkeys; Vaccination; Humoral immunity; Cell mediated immunity; Maternally derived antibodies
16.  Epidermolysa bullosa in Danish Hereford calves is caused by a deletion in LAMC2 gene 
Heritable forms of epidermolysis bullosa (EB) constitute a heterogeneous group of skin disorders of genetic aetiology that are characterised by skin and mucous membrane blistering and ulceration in response to even minor trauma. Here we report the occurrence of EB in three Danish Hereford cattle from one herd.
Two of the animals were necropsied and showed oral mucosal blistering, skin ulcerations and partly loss of horn on the claws. Lesions were histologically characterized by subepidermal blisters and ulcers. Analysis of the family tree indicated that inbreeding and the transmission of a single recessive mutation from a common ancestor could be causative. We performed whole genome sequencing of one affected calf and searched all coding DNA variants. Thereby, we detected a homozygous 2.4 kb deletion encompassing the first exon of the LAMC2 gene, encoding for laminin gamma 2 protein. This loss of function mutation completely removes the start codon of this gene and is therefore predicted to be completely disruptive. The deletion co-segregates with the EB phenotype in the family and absent in normal cattle of various breeds. Verifying the homozygous private variants present in candidate genes allowed us to quickly identify the causative mutation and contribute to the final diagnosis of junctional EB in Hereford cattle.
Our investigation confirms the known role of laminin gamma 2 in EB aetiology and shows the importance of whole genome sequencing in the analysis of rare diseases in livestock.
Electronic supplementary material
The online version of this article (doi:10.1186/s12917-015-0334-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4328060
Cattle; Epidermolysis bullosa; Laminin gamma 2; Hereditary; Congenital; Skin
17.  Assessment into the usage of levetiracetam in a canine epilepsy clinic 
Retrospective studies can complement information derived from double-blinded randomized trials. There are multiple retrospective studies reporting good efficacy and tolerability of the anti-epileptic drug levetiracetam (LEV) in human patients with epilepsy; however, reports of LEV's tolerability and efficacy in dogs with epilepsy remain limited. The purpose of this retrospective study was to describe the use of LEV in a canine epilepsy clinic and determine the long-term efficacy and tolerability of LEV in veterinary clinical practice. The electronic database of a UK based referral hospital was searched for LEV usage in dogs with seizures. Information and data necessary for the evaluation were obtained from a combination of electronic and written hospital records, the referring veterinary surgeons’ records and telephone interviews with dog owners. Only dogs that were reportedly diagnosed with idiopathic epilepsy were included in the study.
Fifty-two dogs were included in this retrospective study. Two treatment protocols were recognised; 29 dogs were treated continuously with LEV and 23 dogs received interval or pulse treatment for cluster seizures. LEV treatment resulted in 69% of dogs having a 50% or greater reduction of seizure frequency whilst 15% of all the dogs were completely free from seizures. Seizure frequency reduced significantly in the whole population. No dog was reported to experience life-threatening side effects. Mild side effects were experienced by 46% of dogs and a significantly higher number of these dogs were in the pulse treatment group. The most common side-effects reported were sedation and ataxia.
LEV appears to be effective and well tolerated for reduction of seizures.
PMCID: PMC4328478
Dog; Safety; Seizure; Tolerability; Treatment
18.  Synovial fluid pharmacokinetics of tulathromycin, gamithromycin and florfenicol after a single subcutaneous dose in cattle 
Deep digital septic conditions represent some of the most refractory causes of severe lameness in cattle. The objective of this study was to determine the distribution of tulathromycin, gamithromycin and florfenicol into the synovial fluid of the metatarsophalangeal (MTP) joint of cattle after single subcutaneous administration of drug to evaluate the potential usefulness of these single-dose, long-acting antimicrobials for treating bacterial infections of the joints in cattle.
Twelve cross-bred beef cows were randomly assigned to one of the drugs. Following subcutaneous administration, arthrocentesis of the left metatarsophalangeal joint was performed at various time points up to 240 hours post-injection, and samples were analyzed for drug concentration. In synovial fluid, florfenicol pharmacokinetic parameters estimates were: mean Tmax 7 +/− 2 hours, mean t½ 64.9 +/− 20.1 hours and mean AUC0-inf 154.0 +/− 26.2 ug*h/mL. Gamithromycin synovial fluid pharmacokinetic parameters estimates were: mean Tmax 8 hours, mean t½ 77.9 +/− 30.0 hours, and AUC0-inf 6.5 +/− 2.9 ug*h/mL. Tulathromycin pharmacokinetic parameters estimates in synovial fluid were: Tmax 19 +/− 10 hours, t½ 109 +/− 53.9 hours, and AUC0-inf 57.6 +/− 28.2 ug h/mL.
In conclusion, synovial fluid concentrations of all three antimicrobials were higher for a longer duration than that of previously reported plasma values. Although clinical data are needed to confirm microbiological efficacy, florfenicol achieved a synovial fluid concentration greater than the MIC90 for F. necrophorum for at least 6 days.
PMCID: PMC4332912
Synovial fluid; Tulathromycin; Gamithromycin; Florfenicol; Pharmacokinetics; Bovine
19.  Mortality associated with avian reovirus infection in a free-living magpie (Pica pica) in Great Britain 
Avian reoviruses (ARVs) cause a range of disease presentations in domestic, captive and free-living bird species. ARVs have been reported as a cause of significant disease and mortality in free-living corvid species in North America and continental Europe. Until this report, there have been no confirmed cases of ARV-associated disease in British wild birds.
Case presentation
Sporadic individual magpie (Pica pica) mortality was detected at a single site in Buckinghamshire, England, April-September 2013. An adult female magpie was found moribund and subsequently died. Post-mortem examination identified hepatomegaly and splenomegaly as the most severe macroscopic abnormalities. Histopathological examination revealed extensive hepatic and splenic necrosis. Transmission electron microscopy (TEM) identified virions of a size (circa 78 nm diameter) and morphology consistent with ARV in both the liver and the small intestinal (SI) contents. Nucleic acid extracted from pooled liver and spleen was positive on both a pan-reovirus nested PCR targeting the RNA-dependent RNA polymerase gene and a PCR using primers specific to the ARV sigma C protein gene. Virus isolated from the liver and the SI contents was characterised by a syncytial-type cytopathic effect, a reovirus-like appearance on TEM and sequence identical to that from PCR of tissues. In situ hybridisation confirmed co-localisation of ARV with lesions in the liver and spleen, implicating ARV as the causative agent. Splenic lymphoid atrophy and necrotic stomatitis associated with Aspergillus fumigatus infection were consistent with generalised immunosuppression and resultant opportunistic infection.
The pathology and comprehensive virus investigations in this case indicate ARV as the primary pathogen in this magpie, with concurrent secondary infection subsequent to immunosuppression, as has been observed with reoviral infections in other bird species. ARV should be considered as a differential diagnosis for magpie, and potentially other corvid, disease and mortality incidents. This is the first demonstration of ARV-associated mortality in a wild bird in Britain. The prevalence and significance of ARV infection in British wild birds, and its implications for poultry and captive bird health, are currently unknown.
PMCID: PMC4336486
Avian reovirus; Magpie; Pica pica; Hepatic necrosis; Splenic necrosis
20.  Cyto-adherence of Mycoplasma mycoides subsp. mycoides to bovine lung epithelial cells 
Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia (CBPP), a respiratory disease of cattle, whereas the closely related Mycoplasma mycoides subsp. capri (Mmc) is a goat pathogen. Cyto-adherence is a crucial step in host colonization by mycoplasmas and subsequent pathogenesis. The aim of this study was to investigate the interactions between Mmm and mammalian host cells by establishing a cyto-adherence flow cytometric assay and comparing tissue and species specificity of Mmm and Mmc strains.
There were little significant differences in the adherence patterns of eight different Mmm strains to adult bovine lung epithelial cells. However, there was statistically significant variation in binding to different host cells types. Highest binding was observed with lung epithelial cells, intermediate binding with endothelial cells and very low binding with fibroblasts, suggesting the presence of effective adherence of Mmm on cells lining the airways of the lung, which is the target organ for this pathogen, possibly by high expression of a specific receptor. However, binding to bovine fetal lung epithelial cells was comparably low; suggesting that the lack of severe pulmonary disease seen in many infected young calves can be explained by reduced expression of a specific receptor.
Mmm bound with high efficiency to adult bovine lung cells and less efficiently to calves or goat lung cells. The data show that cyto-adherence of Mmm is species- and tissue- specific confirming its role in colonization of the target host and subsequent infection and development of CBPP.
Electronic supplementary material
The online version of this article (doi:10.1186/s12917-015-0347-3) contains supplementary material, which is available to authorized users.
PMCID: PMC4336739
Mycoplasma mycoides subsp. mycoides; Contagious bovine pleuropneumonia; Cyto-adherence; Epithelial cells
21.  Hypoxia following etorphine administration in goats (Capra hircus) results more from pulmonary hypertension than from hypoventilation 
Etorphine, a potent opioid agonist, causes pulmonary hypertension and respiratory depression. Whether etorphine-induced pulmonary hypertension negatively influences pulmonary gas exchange and exacerbates the effects of ventilator depression and the resultant hypoxemia is unknown. To determine if these effects occurred we instrumented twelve goats with peripheral and pulmonary arterial catheters to measure systemic and pulmonary pressures before and after etorphine administration. Concurrent cardiopulmonary and arterial blood gas variables were also measured.
Etorphine induced hypoventilation (55% reduction to 7.6 ± 2.7 L.min−1, F(11,44) = 15.2 P < 0.0001), hypoxia (<45 mmHg, F(11,44) = 8.6 P < 0.0001), hypercapnia (>40 mmHg, F(11,44) = 5.6 P < 0.0001) and pulmonary hypertension (mean 23 ± 6 mmHg, F(11,44) = 8.2 P < 0.0001). Within 6 min of etorphine administration hypoxia was twice (F(11,22) = 3.0 P < 0.05) as poor than that expected from etorphine-induced hypoventilation alone. This disparity appeared to result from a decrease in the movement of oxygen (gas exchange) across the alveoli membrane, as revealed by an increase in the P(A-a)O2 gradient (F(11,44) = 7.9 P < 0.0001). The P(A-a)O2 gradient was not correlated with global changes in the ventilation perfusion ratio (P = 0.28) but was correlated positively with the mean pulmonary artery pressure (P = 0.017, r2 = 0.97), indicating that pulmonary pressure played a significant role in altering pulmonary gas exchange.
Attempts to alleviate etorphine-induced hypoxia therefore should focus not only on reversing the opioid-induced respiratory depression, but also on improving gas exchange by preventing etorphine-induced pulmonary hypertension.
PMCID: PMC4322799  PMID: 25644810
Hypoxia; Opioid; Respiratory depression; Oxygen diffusion
22.  Changes in the equine fecal microbiota associated with the use of systemic antimicrobial drugs 
The intestinal tract is a rich and complex environment and its microbiota has been shown to have an important role in health and disease in the host. Several factors can cause disruption of the normal intestinal microbiota, including antimicrobial therapy, which is an important cause of diarrhea in horses. This study aimed to characterize changes in the fecal bacterial populations of healthy horses associated with the administration of frequently used antimicrobial drugs.
Twenty-four adult mares were assigned to receive procaine penicillin intramuscularly (IM), ceftiofur sodium IM, trimethoprim sulfadiazine (TMS) orally or to a control group. Treatment was given for 5 consecutive days and fecal samples were collected before drug administration (Day 1), at the end of treatment (Days 5), and on Days 14 and 30 of the trial. High throughput sequencing of the V4 region of the 16S rRNA gene was performed using an Illumina MiSeq sequencer. Significant changes of population structure and community membership were observed after the use of all drugs. TMS caused the most marked changes on fecal microbiota even at higher taxonomic levels including a significant decrease of richness and diversity. Those changes were mainly due to a drastic decrease of Verrucomicrobia, specifically the “5 genus incertae sedis”. Changes in structure and membership caused by antimicrobial administration were specific for each drug and may be predictable. Twenty-five days after the end of treatment, bacterial profiles were more similar to pre-treatment patterns indicating a recovery from changes caused by antimicrobial administration, but differences were still evident, especially regarding community membership.
The use of systemic antimicrobials leads to changes in the intestinal microbiota, with different and specific responses to different antimicrobials. All antimicrobials tested here had some impact on the microbiota, but TMS significantly reduced bacterial species richness and diversity and had the greatest apparent impact on population structure, specifically targeting members of the Verrucomicrobia phylum.
Electronic supplementary material
The online version of this article (doi:10.1186/s12917-015-0335-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4323147  PMID: 25644524
Horses; Antibiotics; Intestinal microbiota; Intestinal bacteria; Microbiome; Antimicrobial associated diarrhea
23.  Characterisation of recent foot-and-mouth disease viruses from African buffalo (Syncerus caffer) and cattle in Kenya is consistent with independent virus populations 
Understanding the epidemiology of foot-and-mouth disease (FMD), including roles played by different hosts, is essential for improving disease control. The African buffalo (Syncerus caffer) is a reservoir for the SAT serotypes of FMD virus (FMDV). Large buffalo populations commonly intermingle with livestock in Kenya, yet earlier studies have focused on FMD in the domestic livestock, hence the contribution of buffalo to disease in livestock is largely unknown. This study analysed 47 epithelia collected from FMD outbreaks in Kenyan cattle between 2008 and 2012, and 102 probang and serum samples collected from buffalo in three different Kenyan ecosystems; Maasai-Mara (MME) (n = 40), Tsavo (TSE) (n = 33), and Meru (ME) (n = 29).
Antibodies against FMDV non-structural proteins were found in 65 of 102 (64%) sera from buffalo with 44/102 and 53/102 also having neutralising antibodies directed against FMDV SAT 1 and SAT 2, respectively. FMDV RNA was detected in 42% of the buffalo probang samples by RT-qPCR (Cycle Threshold (Ct) ≤32). Two buffalo probang samples were positive by VI and were identified as FMDV SAT 1 and SAT 2 by Ag-ELISA, while the latter assay detected serotypes O (1), A (20), SAT 1 (7) and SAT 2 (19) in the 47 cattle epithelia. VP1 coding sequences were generated for two buffalo and 21 cattle samples. Phylogenetic analyses revealed SAT 1 and SAT 2 virus lineages within buffalo that were distinct from those detected in cattle.
We found that FMDV serotypes O, A, SAT 1 and SAT 2 were circulating among cattle in Kenya and cause disease, but only SAT 1 and SAT 2 viruses were successfully isolated from clinically normal buffalo. The buffalo isolates were genetically distinct from isolates obtained from cattle. Control efforts should focus primarily on reducing FMDV circulation among livestock and limiting interaction with buffalo. Comprehensive studies incorporating additional buffalo viruses are recommended.
Electronic supplementary material
The online version of this article (doi:10.1186/s12917-015-0333-9) contains supplementary material, which is available to authorized users.
PMCID: PMC4334418  PMID: 25644407
African buffalo; Cattle; Control; Epidemiology; Foot-and-mouth disease; Lineages
24.  Hematological and histopathological effects of swainsonine in mouse 
Livestock that consume locoweed exhibit multiple neurological symptoms, including dispirited behavior, staggered gait, trembling, ataxia, impaired reproductive function and cellular vacuolar degeneration of multiple tissues due to toxicity from plant-derived alkaloids such as swainsonine.
Swainsonine was administered to F0 and F1 mice by intraperitoneal injection before, during and after pregnancy at the following doses: 0.525 mg/kg BW(I), 0.2625 mg/kg BW(II), 0.175 mg/kg BW(III) and 0 mg/kg BW(IV). Hemosiderin deposits were observed the lamina propria of endometrium in uterus and the red pulp of spleen. Ovary corpus lutea counts in F0 mice were higher in swainsonine-treated mice compared to control mice. Indirect bilirubin content and reticulocyte numbers were increased in swainsonine-treated F0 and F1 generation mice compared to control group (P < 0.05). Lactate dehydrogenase, alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase content in F0-I and F0-II mice were significantly increased compared with F0-IV group mice (P < 0.05). Red blood cells, hemoglobin and mean corpuscular hemoglobin levels were significantly decreased in F0 and F1 mice compared with the control group (P < 0.05).
Swainsonine exerts effects on estrus period and reproductive ability, and offspring of dams dosed with swainsonine were affected in-utero or from nursing. Damage to liver, uterus and spleen, as well as hematological changes, are observable before neurological symptoms present.
PMCID: PMC4335725  PMID: 25644684
Swainsonine; Locoweed; Hemosiderin deposits; Mouse
25.  Prevalence and genetic characteristics of Salmonella in free-living birds in Poland 
Salmonella species are widespread in the environment, and occur in cattle, pigs, and birds, including poultry and free-living birds. In this study, we determined the occurrence of Salmonella in different wild bird species in Poland, focusing on five Salmonella serovars monitored in poultry by the European Union: Salmonella serovars Enteritidis, Typhimurium, Infantis, Virchow, and Hadar. We characterized their phenotypic and genetic variations.
Isolates were classified into species and subspecies of the genus Salmonella with a polymerase chain reaction (PCR) assay. The prevalence of selected virulence genes (spvB, spiA, pagC, cdtB, msgA, invA, sipB, prgA, spaN, orgA, tolC, ironN, sitC, ipfC, sifA, sopB, and pefA) among the isolated strains was determined. We categorized all the Salmonella ser. Typhimurium strains with enterobacterial repetitive intergenic consensus (ERIC)-PCR.
Sixty-four Salmonella isolates were collected from 235 cloacal swabs, 699 fecal samples, and 66 tissue samples (6.4% of 1000 samples) taken from 40 different species of wild birds in Poland between September 2011 and August 2013. The largest numbers of isolates were collected from Eurasian siskin and greenfinch: 33.3% positive samples for both. The collected strains belonged to one of three Salmonella subspecies: enterica (81.25%), salamae (17.19%), or houtenae (1.56%). Eighteen strains belonged to Salmonella ser. Typhimurium (28.13%), one to ser. Infantis (1.56%), one to ser. Virchow (1.56%), and one to ser. Hadar (1.56%). All isolates contained spiA, msgA, invA, lpfC, and sifA genes; 94.45% of isolates also contained sitC and sopB genes. None of the Salmonella ser. Typhimurium strains contained the cdtB gene. The one Salmonella ser. Hadar strain contained all the tested genes, except spvB and pefA; the one Salmonella ser. Infantis strain contained all the tested genes, except tspvB, pefA, and cdtB; and the one Salmonella ser. Virchow strain contained all the tested genes, except spvB, pefA, cdtB, and tolC.
The Salmonella ser. Typhimurium strains varied across the same host species, but similarity was observed among strains isolated from the same environment (e.g., the same bird feeder or the same lake).
Our results confirm that some wild avian species are reservoirs for Salmonella serotypes, especially Salmonella ser. Typhimurium.
PMCID: PMC4316766  PMID: 25636375
Free-living birds; Salmonella spp; Poland; Virulence genes; ERIC-PCR

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