Comparison of Variable-Number Tandem-Repeat Markers typing and IS1245 Restriction Fragment Length Polymorphism fingerprinting of Mycobacterium avium subsp. hominissuis from human and porcine origins

Background

Animal mycobacterioses are regarded as a potential zoonotic risk and cause economical losses world wide. M. avium subsp. hominissuis is a slow-growing subspecies found in mycobacterial infected humans and pigs and therefore rapid and discriminatory typing methods are needed for epidemiological studies. The genetic similarity of M. avium subsp. hominissuis from human and porcine origins using two different typing methods have not been studied earlier. The objective of this study was to compare the IS1245 RFLP pattern and MIRU-VNTR typing to study the genetic relatedness of M. avium strains isolated from slaughter pigs and humans in Finland with regard to public health aspects.

Methods

A novel PCR-based genotyping method, variable number tandem repeat (VNTR) typing of eight mycobacterial interspersed repetitive units (MIRUs), was evaluated for its ability to characterize Finnish Mycobacterium avium subsp. hominissuis strains isolated from pigs (n = 16) and humans (n = 13) and the results were compared with those obtained by the conventional IS1245 RFLP method.

Results

The MIRU-VNTR results showed a discriminatory index (DI) of 0,92 and the IS1245 RFLP resulted in DI 0,98. The combined DI for both methods was 0,98. The MIRU-VNTR test has the advantages of being simple, reproducible, non-subjective, which makes it suitable for large-scale screening of M. avium strains.

Conclusions

Both typing methods demonstrated a high degree of similarity between the strains of human and porcine origin. The parallel application of the methods adds epidemiological value to the comparison of the strains and their origins. The present approach and results support the hypothesis that there is a common source of M. avium subsp. hominissuis infection for pigs and humans or alternatively one species may be the infective source to the other.     

OCT4 Expression in Outgrowth Colonies Derived from Porcine Inner Cell Masses and Epiblasts

The present study was conducted to test different methods for porcine inner cell mass (ICM) and epiblast isolation and to evaluate the morphology and expression of pluripotency genes in ICM‐ and epiblast‐derived outgrowth colonies (OCs) and passages thereof with particular attention on the relationship between OCT4 expression and embryonic stem cell (ESC)‐like morphology. A total of 104 zona pellucida‐enclosed and 101 hatched blastocysts were subjected to four different methods of ICM and epiblast isolation, respectively: Manual isolation, immunosurgery, immunosurgery with manual cleaning, or whole blastocyst culture. OCs were established on mouse embryonic fibroblast (MEF) cells and categorized according to morphology and OCT4 staining. Although all isolation methods resulted in ESC‐like OCs, immunosurgery with manual cleaning yielded significantly higher rates of ICM/epiblast attachment and subsequent ESC‐like morphology, whereas no significant difference was found between ICM and epiblasts with respect to these characteristics. All ESC‐like OCs showed nuclear OCT4 staining and expression of OCT4, NANOG and SOX2 as evaluated by RT‐PCR. Upon initial passages, the expression of pluripotency markers was, however, gradually lost in spite of maintained ESC‐like morphology. In conclusion, we have established a robust system for derivation of ESC‐like OCs from porcine ICM and epiblasts and we have shown that localization of OCT4 is associated with an ESC‐like morphology although this relationship is lost during early passages.     

A high-performance liquid chromatography method for determination of 4-aminopyridine in tissues and urine

An analytical method was developed to measure 4-aminopyridine in tissues and urine to determine appropriate diagnostic samples in acute poisoning cases. Tissues from rats dosed with 4-aminopyridine were extracted with methylene chloride. Extracts were analyzed by high-performance liquid chromatography using an isocratic solvent system of acetonitrile and aqueous solution (15/85 v/v) consisting of 0.015 M sodium salt of l-heptane-sulfonic acid, 0.002 M tetramethylammonium bromide, and 0.01 M sodium dihydrogen phosphate adjusted to pH 3.0 with phosphoric acid. We concluded that suitable diagnostic samples for acute poisoning cases include stomach contents, kidney, liver, and urine.     

The effect of four anesthetic protocols on the spleen in dogs

Splenic enlargement following administration of barbiturates has been well described in dogs; other agents have not been investigated. This study aimed to compare the effects of four anesthetic protocols on splenic size.
Twenty-four fasted Beagle dogs scheduled for laparotomy were allocated to one of the four groups. Group 1: acepromazine and butorphanol followed by induction with thiopental; Group 2: acepromazine and butorphanol followed by induction with propofol; Group 3: medetomidine and butorphanol followed by induction with propofol; Group 4: medetomidine and butorphanol followed by induction with ketamine and diazepam. Anesthesia was maintained with halothane in oxygen, intravenous fluids were administered. Splenic length, width and height were measured once when the abdomen was opened and again just prior to closure. Spleens were also traced, the image was digitized, and the area was calculated. PCV and total solids were measured before and after pre-medication, after induction, and each time the spleen was measured. Data were analyzed using a Repeated Measures anova with splenic variables indexed by body surface area and dose of induction agent as a covariate.
Area and width of the spleens were less in the dogs of Groups 2 and 3 than in those of the other groups. Splenic area and length did not change significantly during surgery. Dosage of propofol was not significantly different between Groups 2 and 3. Baseline PCV was not significantly different among groups and decreased significantly in all dogs, but at different times. In Groups 1 and 2, the decrease occurred after pre-medication, in Group 3 at induction, and in Group 4 during surgery. A significant decrease in TS occurred in all groups during surgery.
We concluded that the use of propofol resulted in smaller spleen size during surgery than that following the use of thiopental. Multiple factors influenced the PCV.     

Quantification of Mycobacterium avium subspecies in pig tissues by real-time quantitative PCR

Background

Mycobacterioses in animals cause economical losses and certain Mycobacterium avium subspecies are regarded as potential zoonotic agents. The evaluation of the zoonotic risk caused by M. avium subspecies requires information about the quantities of Mycobacterium strains in infected animals. Because M. avium subspecies in pig tissues are difficult or even impossible to quantify by culturing, we tested the suitability of a culture-independent real-time quantitative PCR (qPCR) assay for this purpose.

Methods

Mycobacterial DNA was extracted from porcine tissues by a novel method and quantified by Mycobacterium genus specific qPCR assay targeting the 16S rRNA gene.

Results

The response of the qPCR assay to the amount of M. avium subspecies avium mixed with porcine liver was linear in the range of approximately log10⁵ to log10⁷Mycobacterium cells per 1 g of liver. The assay was validated with three other M. avium subspecies strains. When the assay was applied to porcine lymph nodes with or without visible lesions related to Mycobacterium avium subspecies infections, around 10⁴–10⁷ mycobacterial genomes per gram of lymph nodes were detected.

Conclusions

The qPCR assay was found to be suitable for the quantification of Mycobacterium avium subspecies in porcine lymph nodes and liver.     

Pyloric outflow obstruction secondary to sclerosing encapsulating peritonitis in a dog

A 6-year-old, male neutered mixed breed dog was presented emergently with a three-week history of hyporexia, vomiting, diarrhoea and weight loss. Upon examination, the patient was dull, had generalised muscle atrophy, moderate abdominal pain and a mild amount of peritoneal effusion. A fluid-filled, distended, corrugated small bowel with marked gastroparesis and moderate peritoneal effusion was noted on abdominal ultrasonography. Endoscopy revealed hyperaemic and friable mucosa and a subjectively narrowed pylorus. Emergency exploratory celiotomy was performed due to worsening patient condition and revealed thick, diffuse, fibrous adhesions of the abdominal cavity. Based on these findings, sclerosing encapsulating peritonitis (SEP) was suspected. A large mass of omentum adjacent to the greater curvature of the stomach had caused a pyloric outflow obstruction. Adhesiolysis was attempted but was unsuccessful due to the friability of the small intestines. The dog was humanely euthanased under anaesthesia. A diagnosis of SEP was confirmed via necropsy. No underlying cause was identified. This is the first known case of a pyloric outflow obstruction secondary to SEP in a dog. Although rare, this condition should be considered as a differential for dogs with signs of a pyloric outflow obstruction with concurrent ascites and abdominal pain, hyporexia, vomiting and diarrhoea.     

Evaluation of intraperitoneal and subcutaneous lidocaine and bupivacaine for analgesia following ovariohysterectomy in the dog

The role of ketamine (K) in pain management is controversial. It is reported to provide visceral analgesia in cats. This study aimed to assess its somatic actions using a thermal threshold (TT) model. Six cats (four spayed females, two castrated males, 4.3–7.2 kg) participated in the study. The day before each study, the thorax of each of the cats was shaved and a cephalic catheter was placed. TT was measured using a device specifically developed for cats. A heater element and temperature sensor housed in a small probe were held against the thorax of the cats with an elastic band and pressure bladder to assure consistent contact. The skin temperature was recorded before each test, then the heater was activated. When the cat responded by flinching, turning, or jumping, the stimulus was terminated and the threshold temperature was recorded. Treatments were 2 mg kg^?1 of K (10 mg mL^?1), or 0.2 mL kg^?1 of saline (S) IV, given in a randomized cross‐over design with at least 1 week between treatments. The investigator was blinded to the treatment. TT was measured thrice before treatment (baseline threshold) at 15 minutes, then every 30 minutes for 8 hours and once at 24 hours after injection. Data were analyzed using a four‐factor anova . Cats were sedated for 45 minutes following K treatment. There was no difference in baseline TT between treatments (K = 41.9 ± 1.7 °C, S = 41.0 ± 1.45 °C), and no change in TT at any time in the S group. TT increased significantly at 15 and 30 minutes after K, then decreased below baseline values between 210 and 390 minutes, with a nadir of 38.8 ± ± 1.05 °C at 390 minutes. During this time period, cats exhibited normal activity, but responses to thermal stimuli were exaggerated. This study suggested that K caused a delayed onset hyperalgesia in cats.     

Alfaxalone induction dose following administration of medetomidine and butorphanol in the dog

ObjectiveTo determine in dogs the effects of medetomidine and butorphanol, alone and in combination, on the induction dose of alfaxalone and to describe the induction and intubation conditions.Study designProspective, randomized, blinded clinical trial.AnimalsEighty-five client-owned dogs (ASA 1 or 2).MethodsSubjects were block randomized to treatment group according to temperament. The treatment groups were: medetomidine 4 μg kg^?1 (M), butorphanol 0.1 mg kg^?1 (B), or a combination of both (MB), all administered intramuscularly. After 30 minutes, a sedation score was assigned, and alfaxalone 0.5 mg kg^?1 was administered intravenously over 60 seconds by an observer who was unaware of treatment group. Tracheal intubation conditions were assessed and, if tracheal intubation was not possible after 20 seconds, further boluses of 0.2 mg kg^?1 were given every 20 seconds until intubation was achieved. Induction dose and adverse events (sneezing, twitching, paddling, excitement, apnoea and cyanosis) were recorded; induction quality and intubation conditions were scored and recorded.ResultsThe mean dose of alfaxalone required for induction was similar for groups M and B: 1.2 ± 0.4 mg kg^?1. The mean dose requirement for group MB (0.8 ± 0.3 mg kg^?1) was lower than groups M and B (p < 0.0001). Induction dose was not influenced by temperament or level of sedation. Induction and intubation scores did not differ between treatment groups. Adverse events were noted in 16 dogs; there was no association with treatment group, temperament or level of sedation.Conclusions and clinical relevanceMedetomidine and butorphanol administered in combination reduce the anaesthetic induction dose of alfaxalone compared to either agent alone. This difference should be taken into account when using this combination of drugs in a clinical setting.     

Enantiospecific ketoprofen concentrations in plasma after oral and intramuscular administration in growing pigs

Background

Ketoprofen is a non-steroidal anti-inflammatory drug which has been widely used for domestic animals. Orally administered racemic ketoprofen has been reported to be absorbed well in pigs, and bioavailability was almost complete. The objectives of this study were to analyze R- and S-ketoprofen concentrations in plasma after oral (PO) and intra muscular (IM) routes of administration, and to assess the relative bioavailability of racemic ketoprofen for both enantiomers between those routes of administration in growing pigs.

Methods

Eleven pigs received racemic ketoprofen at dose rates of 4 mg/kg PO and 3 mg/kg IM in a randomized, crossover design with a 6-day washout period. Enantiomers were separated on a chiral column and their concentrations were determined by liquid chromatography-tandem mass spectrometry. Pharmacokinetic parameters were calculated and relative bioavailability (F_rel) was determined for S and R –ketoprofen.

Results

S-ketoprofen was the predominant enantiomer in pig plasma after administration of the racemic mixture via both routes. The mean (± SD) maximum S-ketoprofen concentration in plasma (7.42 mg/L ± 2.35 in PO and 7.32 mg/L ± 0.75 in IM) was more than twice as high as that of R-ketoprofen (2.55 mg/L ± 0.99 in PO and 3.23 mg/L ± 0.70 in IM), and the terminal half-life was three times longer for S-ketoprofen (3.40 h ± 0.91 in PO and 2.89 h ± 0.85 in IM) than R-ketoprofen (1.1 h ± 0.90 in PO and 0.75 h ± 0.48 in IM). The mean (± SD) relative bioavailability (PO compared to IM) was 83 ± 20% and 63 ± 23% for S-ketoprofen and R-ketoprofen, respectively.

Conclusions

Although some minor differences were detected in the ketoprofen enantiomer concentrations in plasma after PO and IM administration, they are probably not relevant in clinical use. Thus, the pharmacological effects of racemic ketoprofen should be comparable after intramuscular and oral routes of administration in growing pigs.     

Sensitivity and specificity of serum copper determination for detection of copper deficiency in feeder calves

OBJECTIVE: To determine the relationship between serum and liver copper concentrations and evaluate serum copper determination for diagnosis of copper deficiency in juvenile beef calves. DESIGN: Cross-sectional study. ANIMALS: 105 juvenile beef calves. PROCEDURE: Copper concentrations were measured in paired liver and serum samples from 6- to 9-month-old beef calves. Regression models that predicted liver copper concentration as a function of serum copper concentration were developed. Sensitivity and specificity of serum copper concentration for detection of low liver copper concentration were determined, using a range of serum copper concentrations as test endpoints. Positive and negative predictive values were calculated. RESULTS: The association between serum and liver copper concentrations was significant; however, regression models accounted for only a small portion of the variation in liver copper concentrations. For a serum copper concentration endpoint of 0.45 microg/g, sensitivity and specificity for detection of low liver copper concentration were 0.53 and 0.89, respectively. Positive and negative predictive values of serum copper concentration for detection of low liver copper concentration ranged from 0.37 to 0.85 and 0.63 to 0.94, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Regression models are inappropriate for predicting copper status as a function of serum copper concentration. Serum copper concentration is fairly specific for detection of low liver copper concentration but only marginally sensitive when serum copper concentration of 0.45 microg/g is used as a test endpoint. The value of serum copper concentration as a diagnostic indicator depends on prevalence of copper deficiency.