Development of a diagnostic PCR assay based on novel DNA sequences for the detection of Mycoplasma suis (Eperythrozoon suis) in porcine blood

An efficient method of control of porcine eperythrozoonosis (PE) caused by Mycoplasma suis is eradication of infection by detection and removal of infected carrier animals. At present, only a few tests are available for the diagnosis of these latent M. suis infections in pigs. The objective of this study was to develop a PCR assay based on novel DNA sequences for the identification of M. suis-infected pigs. A 1.8 kb EcoRI DNA fragment of the M. suis genome was isolated from the blood of pigs experimentally infected with M. suis. Specificity of the DNA fragment was confirmed by DNA sequence analysis and PCR using primers directed against sequences contained in the 1.8 kb fragment. PCR products of 782 bp in size were amplified only from M. suis particles prepared from the blood of experimentally infected pigs but not from any controls, comprising blood from gnotobiotic piglets and a panel of bacteria including other porcine mycoplasmas. PCR results were confirmed by dot blot hybridisation. The applicability of the PCR assay to diagnose M. suis infections in pigs was evaluated by investigating blood samples from 10 symptomatic pigs with clinical signs typical of porcine eperythrozoonosis and blood samples from 10 healthy pigs. The M. suis-specific PCR product was amplified from all samples taken at episodes of acute disease as well as from samples taken during the latent stage of infection, thus demonstrating the suitability of the PCR assay for detecting latent infected carrier animals.     

Evaluation of acetaminophen absorption in horses with experimentally induced delayed gastric emptying

OBJECTIVE: To evaluate the correlation between the half-time of liquid-phase gastric emptying (T50) determined by use of nuclear scintigraphy, using technetiumTc 99m pentetate, and absorption variables of orally administered acetaminophen in horses with experimentally delayed gastric emptying. ANIMALS: 6 mature horses. PROCEDURE: Delayed gastric emptying was induced by IV injection of atropine sulfate. Twenty minutes later, acetaminophen and technetium Tc 99m pentetate were administered simultaneously via nasogastric tube. Serial lateral images of the stomach region were obtained, using a gamma camera. Power exponential curves were used for estimation of T50 and modified R2 values for estimation of goodness-of-fit of the data. Serial serum samples were obtained, and acetaminophen concentration was determined, using fluorescence polarization immunoassay. Maximum serum concentration (Cmax), time to reach maximum serum concentration (Tmax), area under the curve for 480 minutes, and the appearance rate constant were determined, using a parameter estimation program. Correlations were calculated, using a Spearman rank correlation coefficient. RESULTS: A significant correlation was detected between T50 determined by use of scintigraphy and Tmax determined by use of acetaminophen absorption. Correlation between T50 and other absorption variables of acetaminophen was not significant. CONCLUSIONS AND CLINICAL RELEVANCE: The acetaminophen absorption method was a valid technique in this model of delayed gastric emptying in horses. The method may be a valuable tool for use in research as well as in clinical evaluation of gastric emptying in horses.     

How to substantiate eradication of bovine brucellosis when aspecific serological reactions occur in the course of brucellosis testing

Collaborative work was financed by the EU to develop and assess new diagnostic tools that can differentiate between bovine brucellosis and bovine infections due to Yersinia enterocolitica O:9 either in conjunction with, or as an alternative to, the classical serological, bacteriological or allergic skin tests. Sixteen heifers were experimentally infected with Brucella abortus biovar 1 (five heifers), Brucella suis biovar 2 (two heifers), Y. enterocolitica O:9 (six heifers) and Y. enterocolitica O:3 (three heifers). Four heifers, naturally infected with Y. enterocolitica O:9 that presented aspecific brucellosis serological reactions were also included in the experiment. A self-limited infection was induced in cattle by B. suis biovar 2. All the brucellosis serological tests used, i.e. the slow agglutination test (SAW), the Rose Bengal test (RB), the complement fixation test (CFT), indirect and competitive ELISA’s, lacked specificity when used to analyze sera from Y. enterocolitica O:9 infected animals. A Yersinia outer membrane proteins (YOPs)-ELISA was also used and although the test is able to detect a Yersinia group infection, it provided no evidence of whether or not there is a possible brucellosis infection when dual infections are present. The brucellergen IFN-γ test showed a lack of specificity also. The only test that was proven to be specific is the brucellergen skin test. All brucellosis serological tests, except the indirect ELISA, were limited in their ability to detect B. abortus persistently infected animals.

Based on these experimental studies, a strategy was implemented as part of the year 2001 Belgian Brucellosis Eradication Program to substantiate the eradication of bovine brucellosis. Epidemiological inquiries have identified risk factors associated with aspecific serological reactions, possible transmission and infection of cattle by B. suis biovar 2 from infected wild boars; and both legal and administrative measures taken by the veterinary services. No cases of bovine brucellosis have been confirmed in Belgium since March 2000.     

Storage factors control germination and seedling establishment of freshly harvested true potato seed

Freshly harvested true potato(Solanum tuberosum subsp.andigena, Juz. et Buk.) seeds of the ancient Peruvian cultivar Ccompis were dried to 3.4, 4.2,5.1,6.1, and 7.3% moisture content (Dwt basis) and hermetically stored for 6 months at 15, 30, and 45C. Seeds were humidified to uniform moisture (13%) prior to monthly evaluations of germination at 27 and 17C and seedling performance under variable soil temperature. The effects of treatments on percentage of germination after 8 days at 27C were highly correlated with percentage of emergence at 10 days after sowing and foliage dry weight at 17 days. Germination at 27C increased from negligible to <90% with progressive increases in moisture, temperature, and time in storage. However, germination decreased within the first month of storing seeds at 45C with >5.1% moisture, and after 3 months seeds failed to germinate even after 8 days of lowering the temperature in the germination environment to 17C. Otherwise, germination inhibition at 27C was attributed to dormancy since most seeds readily germinated at 17C. Dormancy was sufficiently released and optimum overall performance was obtained after 2 months of storing seeds at 45C and 4.2% moisture.     

Effect of vaccination on fecal cortisol metabolites in cats and dogs

The aim of this study was to apply a cortisol metabolite determination in the faeces of cats and dogs for monitoring disturbances. In this experiment faeces from every spontaneous defecation of 10 cats and 10 dogs (5 of each sex) were collected starting from one day before until two days after the yearly vaccination. Concentrations of 11,17-dioxoandrostanes (cat) and cortisol equivalents (dog) were determined by enzyme immunoassay (EIA). Faecal cortisol metabolites increased and reached peak concentrations (median: 412% in cats and 417% in dogs, respectively above baseline values) in one of the next two samples following the vaccination. This indicated an activation of the adrenocortex, the degree to which the different parts (physical and psychological components) of the whole vaccination procedure contributed to it was not evaluated. From this experiment we conclude that measuring cortisol metabolites in the faeces is a non-invasive method to monitor stressful conditions in cats and dogs and thus is a valuable tool for evaluating animal welfare.     

Standing laparoscopic herniorrhaphy in stallions using cylindrical polypropylene mesh prosthesis

Standing laparoscopic herniorrhaphy was performed in 9 stallions. Appropriate analgesia was achieved by sedation with detomidine and local flank infiltration with mepivacaine. Three portal sites at the paralumbar fossa were used to perform the herniorrhaphy by means of triangulation. A cylindrical polypropylene mesh was inserted and fixated in the inguinal canal. Subsequent adhesion formation resulted in an obliterated inguinal canal within 2 weeks. This minimal invasive technique allowed us to perform a testis sparing herniorrhaphy in the standing horse.     

Atypical/Nor98 scrapie: properties of the agent, genetics, and epidemiology

Atypical/Nor98 scrapie cases in sheep were diagnosed for the first time in Norway in 1998. They are now identified in small ruminants in most European countries and represent an increasingly large proportion of the scrapie cases diagnosed in Europe. Atypical/Nor98 scrapie isolates have shown to be experimentally transmissible into transgenic mice and sheep but the properties of the TSE agent involved, like its biological and biochemical features, are so clearly distinct from the agent involved in classical scrapie that they have provided a challenging diagnostic for many years. No strain diversity has yet been identified among the atypical/Nor98 scrapie sample cases. The genetic predisposition of the sheep affected by atypical/Nor98 scrapie is almost inverted compared to classical scrapie, and the exact origin of this sporadic TSE strain is still speculative, but a spontaneous, non-contagious origin, like sporadic Creutzfeldt-Jakob disease in humans, can not be excluded. Further transmission and epidemiological studies are needed to better address this hypothesis.     

Prevalence of antimicrobial resistance among bacterial pathogens isolated from cattle in different European countries: 2002–2004

Background

The project "Antibiotic resistance in bacteria of animal origin – II" (ARBAO-II) was funded by the European Union (FAIR5-QLK2-2002-01146) for the period 2003–2005, with the aim to establish a continuous monitoring of antimicrobial susceptibility among veterinary laboratories in European countries based on validated and harmonised methodologies. Available summary data of the susceptibility testing of the bacterial pathogens from the different laboratories were collected.

Method

Antimicrobial susceptibility data for several bovine pathogens were obtained over a three year period (2002–2004). Each year the participating laboratories were requested to fill in excel-file templates with national summary data on the occurrence of antimicrobial resistance from different bacterial species.A proficiency test (EQAS – external quality assurance system) for antimicrobial susceptibility testing was conducted each year to test the accuracy of antimicrobial susceptibility testing in the participating laboratories. The data from this testing demonstrated that for the species included in the EQAS the results are comparable between countries.

Results

Data from 25,241 isolates were collected from 13 European countries. For Staphylococcus aureus from bovine mastitis major differences were apparent in the occurrence of resistance between countries and between the different antimicrobial agents tested. The highest frequency of resistance was observed for penicillin. For Mannheimia haemolytica resistance to ampicillin, tetracycline and trimethoprim/sulphonamide were observed in France, the Netherlands and Portugal. All isolates of Pasteurella multocida isolated in Finland and most of those from Denmark, England (and Wales), Italy and Sweden were susceptible to the majority of the antimicrobials. Streptococcus dysgalactiae and Streptococcus uberis isolates from Sweden were fully susceptible. For the other countries some resistance was observed to tetracycline, gentamicin and erythromycin. More resistance and variation of the resistance levels between countries were observed for Escherichia coli compared to the other bacterial species investigated.

Conclusion

In general, isolates from Denmark, England (and Wales), the Netherlands, Norway, Sweden and Switzerland showed low frequencies of resistance, whereas many isolates from Belgium, France, Italy, Latvia and Spain were resistant to most antimicrobials tested. In the future, data on the prevalence of resistance should be used to develop guidelines for appropriate antimicrobial use in veterinary medicine.     

Occurrence of antimicrobial resistance among bacterial pathogens and indicator bacteria in pigs in different European countries from year 2002 – 2004: the ARBAO-II study

Background

The project "Antibiotic resistance in bacteria of animal origin – II" (ARBAO-II) was funded by the European Union (FAIR5-QLK2-2002-01146) for the period 2003–05. The aim of this project was to establish a program for the continuous monitoring of antimicrobial susceptibility of pathogenic and indicator bacteria from food animals using validated and harmonised methodologies. In this report the first data on the occurrence of antimicrobial resistance among bacteria causing infections in pigs are reported.

Methods

Susceptibility data from 17,642 isolates of pathogens and indicator bacteria including Actinobacillus pleuropneumoniae, Streptococcus suis and Escherichia coli isolated from pigs were collected from fifteen European countries in 2002–2004.

Results

Data for A. pleuropneumoniae from infected pigs were submitted from five countries. Most of the isolates from Denmark were susceptible to all drugs tested with the exceptions of a low frequency of resistance to tetracycline and trimethoprim – sulphonamide.Data for S. suis were obtained from six countries. In general, a high level of resistance to tetracycline (48.0 – 92.0%) and erythromycin (29.1 – 75.0%) was observed in all countries whereas the level of resistance to ciprofloxacin and penicillin differed between the reporting countries. Isolates from England (and Wales), France and The Netherlands were all susceptible to penicillin. In contrast the proportion of strains resistant to ciprofloxacin ranged from 12.6 to 79.0% (2004) and to penicillin from 8.1 – 13.0% (2004) in Poland and Portugal.Data for E. coli from infected and healthy pigs were obtained from eleven countries. The data reveal a high level of resistance to tetracyclines, streptomycin and ampicillin among infected pigs whereas in healthy pigs the frequency of resistance was lower.

Conclusion

Bacterial resistance to some antimicrobials was frequent with different levels of resistance being observed to several antimicrobial agents in different countries. The occurrence of resistance varied distinctly between isolates from healthy and diseased pigs, with the isolates from healthy pigs generally showing a lower level of resistance than those from diseased pigs.The study suggests that the choice of antimicrobials used for the treatment of diseased animals should preferably be based on knowledge of the local pattern of resistance.