Use of a quantitative TaqMan-PCR for the fast quantification of mycobacteria in broth culture, eukaryotic cell culture and tissue

The quantification of slow-growing mycobacteria such as Mycobacterium tuberculosis or M. bovis from in vitro and in vivo samples is complicated by their long generation time, their ability to form aggregates, and their capacity to persist in a state of dormancy. We compared different methods for the establishment of growth curves for broth cultures of M. bovis bacille Calmette-Guérin (BCG). A quantitative TaqMan-PCR yielded results comparable with those obtained by protein quantification and measurement of the ATP content of the cultures. The quantitative TaqMan-PCR furthermore turned out to be particularly suitable for the measurement of multiplication of BCG within eukaryotic cells. Furthermore, it is a fast method allowing an estimation of the mycobacterial load in tissue long before colony counts can be obtained.     

Automated plasmapheresis compared with other plasma collection methods in the horse

The purpose of this study was to evaluate plasmapheresis as a method for plasma extraction in comparison with centrifugation or gravity sedimentation. The study was designed as a cross over trial with six Freiberger horses undergoing plasma donation by plasmapheresis followed by whole-blood donation and subsequent plasma production 4 weeks later. Automated plasmapheresis and whole-blood donation were well tolerated in all horses. The plasmapheresis method achieved an almost complete removal of erythrocytes and leucocytes from plasma at all flow rates. After blood bag centrifugation, significantly more erythrocytes (P < 0.01) and leucocytes (P < 0.01) were present in the prepared plasma compared with plasmapheresis plasma. Plasma prepared by gravity sedimentation contained significantly more erythrocytes and leucocytes than plasma prepared with the other techniques (P < 0.01), and platelet aggregation was observed. The coagulation proteins and the total protein content of plasma prepared after plasmapheresis and blood bag centrifugation did not differ significantly from values measured prior to collection (P > 0.05). However, the activity of factor VIII was significantly lower 24 h after gravity sedimentation of blood than activity prior to blood collection (P < 0.01). In conclusion, automated plasmapheresis is the method of choice for the production of high quality equine plasma.     

Changes in coagulation and markers of fibrinolysis in horses undergoing colic surgery

Activation of coagulation can be frequently found in horses with colic. However, it has also been demonstrated as a sequela of surgical trauma alone in humans. The purpose of the present study was to determine changes in coagulation and fibrinolysis in horses that underwent colic surgery and to evaluate whether these changes were secondary to the colic or the surgery and wound healing. Thirty horses that underwent colic surgery with uncomplicated recovery were included. Ten horses with a Forssell's procedure served as control group with a standardized surgical trauma. Besides daily physical examinations during the observation period of 10 days, activated partial thromboplastin time (aPTT), prothrombin time and thrombin time as well as fibrin monomer (FM), D-Dimer (DD) and antithrombin (AT) III were determined. Compared with the control group the aPTT was the only standard coagulation test that was significantly prolonged before and after the event of colic surgery. After surgery, hyperfibrinogenaemia occurred in all groups. In colic groups FM and DD concentrations were within reference range at admission,and were significantly greater than in control horses after surgery. AT III activity decreased after colic surgery, but did not change in the control group. It was concluded that an activated coagulation state after colic surgery has to be expected, resulting not only from the colic disease, but also from the event of surgery.     

Metastatic gastric adenocarcinoma and diffuse hyperplastic gastritis resembling human Menétrier's disease in a camel (Camelus ferus bactrianus)

A captive 16-year-old male camel (Camelus ferus bactrianus) was euthanized after a prolonged period of inappetence leading to cachexia. At necropsy, there was a 7 cm large, tan, firm, well-demarcated nodule in the tunica muscularis and serosa of the distal region of C3. Histologically, a gastric adenocarcinoma was diagnosed. Numerous metastases were found in the liver and the hepatic lymph nodes, in the wall of the portal vein and the aorta, in the lung, heart, and pleura parietalis. Osseous metaplasia was found within the pleural and aortic metastases. In the mucosa of the glandular region of the C3 compartment a diffuse marked hypertrophy of rugae resembling cerebral convolutions was observed. The lesion was characterized by glandular hyperplasia and stromal inflammation and oedema. These changes closely resembled Menétrier's disease described in humans. To our knowledge, this is the first report of concomitant metastatic gastric adenocarcinoma and gastric hyperplasia in a camel.     

Intramammary infusion of beta1,3-glucan for prevention and treatment of Staphylococcus aureus mastitis

Udder health problems associated with Staphylococcus aureus infections in dairy cows are difficult to control and antibiotics have limited effects. Lately, more interest has been directed towards ways to stimulate the innate immune mechanisms of the animal for better prevention and treatment of mastitis. The objectives of this study were to investigate if intramammary infusion at drying off with the immune modulator beta1,3-glucan can make the udder more resistant to experimental intra mammary S. aureus infection at this time, and to study if intramammary infusion of beta1,3-glucan into lactating udder quarters with chronic subclinical S. aureus infection can stimulate the clearing of the infection. Another aim was to evaluate the effect of beta1,3-glucan on the expression of major histocompatibility complex (MHC class II) on mammary leucocytes, measured by flow cytometry, during these circumstances. The results indicated a slight, but not statistically significant, positive effect of beta1,3-glucan at drying off on the clinical and anti-bacterial response to S. aureus infection, but no therapeutic effect of beta1,3-glucan treatment of udder quarters with chronic subclinical S. aureus mastitis. However, the proportion of MHCII+ milk lymphocytes tended to increase after glucan infusion in those udder quarters indicating a stimulation of the antigen presenting ability. To further evaluate a possible preventive effect of beta1,3-glucan infusion at drying off more studies are needed involving a larger number of animals.     

Analysis of genotypic diversity data for populations of microorganisms

ABSTRACT Estimation of genotypic diversity is an important component of the analysis of the genetic structure of plant pathogen and microbial populations. Estimates of genotypic diversity are a function of both the number of genotypes observed in a sample (genotype richness) and the evenness of distribution of genotypes within the sample. Currently used measures of genotypic diversity have inherent problems that could lead to incorrect conclusions, particularly when diversity is low or sample sizes differ. The number of genotypes observed in a sample depends on the technique used to assay for genetic variation; each technique will affect the maximum number of genotypes that can be detected. We developed an approach to analysis of genotypic diversity in plant pathology that makes specific reference to the techniques used for identifying genotypes. Preferably, populations that are being compared should be very similar in sample size. In this case, the number of genotypes observed can be used directly for comparing richness. In most cases, sample sizes differ and use of the rarefaction method to calculate richness is more appropriate. In all cases, scaling either Stoddart and Taylor's G or Shannon and Wiener's H' by sample size should be avoided. Under those circumstances where it might be important to distinguish whether richness or evenness contribute more to diversity, a bootstrapping approach, where confidence intervals are calculated for indices of diversity and evenness, is recommended.     

Serum concentrations of 1,25-dihydroxycholecalciferol and 25-hydroxycholecalciferol in clinically normal dogs and dogs with acute and chronic renal failure

OBJECTIVE: To compare serum concentrations of 1,25-dihydroxycholecalciferol (1,25-[OH]2D3) and 25-hydroxycholecalciferol (25-[OH]D3) in healthy control dogs and dogs with naturally occurring acute renal failure (ARF) and chronic renal failure (CRF). ANIMALS: 24 control dogs, 10 dogs with ARF, and 40 dogs with CRF. PROCEDURE: Serum concentrations of 1,25-(OH)2D3 were measured by use of a quantitative radioimmunoassay, and serum concentrations of 25-(OH)D3 were measured by use of a protein-binding assay. RESULTS: Mean +/- SD serum concentration of 1,25-(OH)2D3 was 153 +/- 50 pmol/L in control dogs, 75 +/- 25 pmol/L in dogs with ARF, and 93 +/- 67 pmol/L in dogs with CRF. The concentration of 1,25-(OH)2D3 did not differ significantly between dogs with ARF and those with CRF and was in the reference range in most dogs; however, the concentration was significantly lower in dogs with ARF or CRF, compared with the concentration in control dogs. Mean +/- SD concentration of 25-(OH)D3 was 267 +/- 97 nmol/L in control dogs, 130 +/- 82 nmol/L in dogs with ARF, and 84 +/- 60 nmol/L in dogs with CRF. The concentration of 25-(OH)D3 was significantly lower in dogs with ARF or CRF, compared with the concentration in control dogs. CONCLUSIONS AND CLINICAL RELEVANCE: The concentration of 1,25-(OH)2D3 was within the reference range in most dogs with renal failure. Increased serum concentrations of parathyroid hormone indicated a relative deficiency of 1,25-(OH)2D3. A decrease in the serum concentration of 25-(OH)D3 in dogs with CRF appeared to be attributable to reduced intake and increased urinary loss.     

Cell-specific activation of aflatoxin B1 correlates with presence of some cytochrome P450 enzymes in olfactory and respiratory tissues in horse

Horses may be exposed to aflatoxin B(1) (AFB(1)) via inhalation of mouldy dust, leading to high exposure of olfactory and respiratory tissues. In the present study the metabolic activation of AFB(1) was examined in olfactory and respiratory tissues in horse. The results showed covalent binding of AFB(1)-metabolites in sustentacular cells and cells of Bowman's glands in the olfactory mucosa, in some cells of the surface epithelium of nasal respiratory, tracheal, bronchial and bronchiolar mucosa and in some glands in these areas. Immunohistochemistry revealed that cells expressing proteins reacting with CYP 3A4- and CYP 2A6/2B6-antibodies had a similar distribution as those having capacity to activate AFB(1). Our data indicate that the cell-specific activation of AFB(1) correlates with presence of some CYP-enzymes in olfactory and respiratory tissues in horse.     

Use and reliability of the Delvo-P-test

The Delvo-P-test (Gist-brocades nr, Delft, The Netherlands) is based on biological detection of antibiotics in the milk. According to the procedure of the manufacture the test is suited for bulk milk processing factories. Problems arise when the test is used on milk samples of a single cow. Buffers, cells, bacteria, cell contents, and desinfection without using antibiotics may give false positive and false negative results. An error up to 44.8% was detected on single cow milk samples. The test is well suited for bulk milk, where the processing of milk follows a biological procedure as for cheese. The use on single cow milk samples to prove absence of antibiotics is not recommended.