Veterinary Samples Research Articles

Characterization of Bromethalin and its Degradation Products in Veterinary Toxicology Samples by GC-MS-MS.

Bromethalin is a neurotoxicant with unusual instability and chromatographic behavior that make it difficult to analyze by gas chromatography (GC) in forensic examination of non-target animal deaths. Physicochemical breakdown of bromethalin produced multiple unique products with discernible mass spectra. This paper describes an investigation of the GC electron impact-mass spectrometric properties of bromethalin and its capacity to generate up to twenty heat- or light-induced breakdown products. Two principal breakdown products are isomeric with one another and involve release of both fluorine and methyl groups to develop dehydrofluorodesmethylbromethalin products. These compounds have proven to be excellent surrogate markers in screening forensic samples for bromethalin exposure, particularly in veterinary samples in which the active metabolite desmethylbromethalin has not yet accumulated to any appreciable extent, such as baits and animal stomach contents. The compounds as well as their parent bromethalin were easily monitored by GC interfaced with a tandem-quadrupole mass spectrometer using multiple-reaction monitoring (MRM) modes. Unusual gas chromatographic properties of bromethalin included: (i) specific requirements for a maximum oven temperature; (ii) non-linear increases in detector response on increased injection volumes, hypothesized to result from variable diffusion coefficients. We report here the development of GC strategies that facilitate detection of bromethalin and its breakdown products, as well as their MRM analysis by tandem-quadrupole mass spectrometry. The developed approaches are applicable to feed, baits and stomach contents as well as extracted tissue samples such as liver and kidney.

PCR-terminal restriction fragment length polymorphism for direct detection and identification of dermatophytes in veterinary mycology

The biological diagnosis of dermatophytosis in veterinary medicine usually relies on direct microscopic examination and inoculation of the samples on appropriate culture media. However, identification of dermatophytes needs expertise, and cultures which require from days to weeks to be conclusive, may lack of sensitivity because of the quite common overgrowth of contaminants. Here we developed a polymerase chain reaction (PCR) assay based on terminal restriction fragment length polymorphism (TRFLP), which may improve sensitivity of the biological diagnosis and reduce the delay for initiation of treatment. This study was first conducted on pure cultures of various dermatophytes (27 species), yeasts (14 species) and moulds (45 species). After DNA extraction, the internal transcribed spacer (ITS)-28S region of ribosomal DNA was amplified with primers targeting specifically pathogenic dermatophytes, and species of interest were identified by TRFLP with appropriate restriction enzymes. After validation, this assay was applied to veterinary samples and results were compared to those obtained by direct microscopic examination and cultures. All target species were correctly identified, and none of the yeast or mould species was amplified, demonstrating specificity of the assay. Regarding clinical samples, the causative agent was detected by PCR-TRFLP from 97.1% of the samples with both positive direct microscopic examination and cultures. No dermatophytes were detected when both conventional tests were negative. PCR-TRFLP developed here demonstrated to be highly sensitive and specific, allowing rapid detection and direct identification of dermatophytes in veterinary practice. Therefore, this assay is especially suitable for the biological diagnosis of dermatophytosis in different animal species.

Virus classification \u2013 where do you draw the line?

High-throughput sequencing (HTS) and its use in recovering and assembling novel virus sequences from environmental, human clinical, veterinary and plant samples has unearthed a vast new catalogue of viruses. Their classification, known by their sequences alone, sets a major challenge to traditional virus taxonomy, especially at the family and species levels, which have been historically based largely on descriptive taxon definitions. These typically entail some knowledge of their phenotypic properties, including replication strategies, virion structure and clinical and epidemiological features, such as host range, geographical distribution and disease outcomes. Little to no information on these attributes is available, however, for viruses identified in metagenomic datasets. If such viruses are to be included in virus taxonomy, their assignments will have to be guided largely or entirely by metrics of genetic relatedness. The immediate problem here is that the International Committee on Taxonomy of Viruses (ICTV), an organisation that authorises the taxonomic classification of viruses, provides little or no guidance on how similar or how divergent viruses must be in order to be considered members of new species or new families. We have recently developed a method for scoring genomic (dis)similarity between viruses (Genome Relationships Applied to Virus Taxonomy – GRAViTy) among the eukaryotic and prokaryotic viruses currently classified by the ICTV. At the family and genus levels, we found large-scale consistency between genetic relationships and their taxonomic assignments for eukaryotic viruses of all genome configurations and genome sizes. Family assignments of prokaryotic viruses have, however, been made at a quite different genetic level, and groupings currently classified as sub-families are a much better match to the eukaryotic virus family level. These findings support the ongoing reorganisation of bacteriophage taxonomy by the ICTV Phage Study Group. A rapid and objective means to explore metagenomic viral diversity and make evidence-based assignments for such viruses at each taxonomic layer is essential. Analysis of sequences by GRAViTy provides evidence that family (and genus) assignments of currently classified viruses are largely underpinned by genomic relatedness, and these features could serve as a guide towards an evidence-based classification of metagenomic viruses in the future.

Prevalence of Group I Salmonella Kentucky in domestic food animals from Pennsylvania and overlap with human clinical CRISPR sequence types.

Although infrequently associated with illness in humans, Salmonella enterica, subsp. enterica serovar Kentucky is the most common non-clinical, non-human serovar reported in the United States, being largely found in poultry and poultry products, as well as being associated with cattle. This serovar is polyphyletic and can be separated into two groups, Group I and II, based on CRISPR-typing analysis. In Salmonella Kentucky isolates from human clinical samples in Pennsylvania, both lineages are equally represented. The goal of this study was to determine whether both groups were also represented in domestic food animals in Pennsylvania. We analysed the CRISPR arrays from 67 Salmonella Kentucky isolates used PCR and sequencing of CRISPR arrays or analysis of whole genome sequences to analyse the CRISPR arrays and Across a collection of 67 Salmonella Kentucky isolates that includes those collected from farms, veterinary clinical samples as well as isolates from retail meats, we show that Group I Salmonella Kentucky are the exclusive lineage present. We reveal that the specific subtype of over a quarter of these animal isolates are also found to be responsible for causing human salmonellosis in the same region over the same time period.

National outbreak of Salmonella Give linked to a local food manufacturer in Malta, October 2016.

Salmonella Give is a rare serotype across Europe. In October 2016, a national outbreak of S. Give occurred in Malta. We describe the epidemiological, environmental, microbiological and veterinary investigations. Whole-genome sequencing (WGS) was performed on human, food, environmental and veterinary isolates. Thirty-six human cases were reported between October and November 2016, 10 (28%) of whom required hospitalisation. Twenty-six (72%) cases were linked to four restaurants. S. Give was isolated from ready-to-eat antipasti served by three restaurants which were all supplied by the same local food manufacturer. Food-trace-back investigations identified S. Give in packaged bean dips, ham, pork and an asymptomatic food handler at the manufacturer; inspections found inadequate separation between raw and ready-to-eat food during processing. WGS indicated two genetically distinguishable strains of S. Give with two distinct clusters identified; one cluster linked to the local food manufacturer and a second linked to veterinary samples. Epidemiological, environmental and WGS evidence pointed towards cross-contamination of raw and ready-to-eat foods at the local manufacturer as the likely source of one cluster. Severity of illness indicates a high virulence of this specific serotype. To prevent future cases and outbreaks, adherence to food safety practices at manufacturing level need to be reinforced.

Multi-Walled Carbon Nanotubes as Efficient Sorbent for the Solid Bar Microextraction of non-Steroidal Anti-Inflammatory Drugs from Human Urine Samples

Background: The determination of NSAIDs (ketoprofen, diclofenac, and ibuprofen) in urine samples provides useful information for assessing their safety, therapeutic effect, and their mechanism of action. Urine samples are characterized by their complexity and low concentration of the target analytes, which make their direct analysis difficult. In this work, the potential of multi-walled carbon nanotubes (MWCNTs) as Solid Bar Microextraction (SBME) adsorbents for extraction and preconcentration of selected drugs from urine samples have been investigated. Methods: Five SBME devices (contains 10 mg of MWCNTs) were placed in 30 mL of adjusted pH 2 water samples and stirred for 40 minutes. After finishing the extraction, the devices were ultrasonicated in 250 µL of methanol for 5 min to desorb the selected drugs and analyzed using HPLC-DAD. Results: The analytical performance of the whole method was evaluated in water samples. Limits of detection and quantitation were in the ranges of (0.36- 0.52) and (1.2- 1.7) µg L-1, respectively, and the Relative Standard Deviation (RSD) < 5.7% over a concentration range of 5-100 µg L-1. Application: The proposed method was successfully applied to the analysis of selected drugs in spiked human urine samples. The absolute recovery obtained by spiking the urine samples at three concentration levels: 5, 50 and 100 µg L-1 of selected NSAIDs were from 48.8% to 53.5%. Conclusion: The proposed SBME using MWCNT as a sorbent material can be a useful alternative sample preparation technique for the routine determination of NSAIDs in urine samples at therapeutic levels with very simple sample pretreatment. Furthermore, the application of the developed SBME method might be extended to study the determination of selected non-steroidal anti-inflammatory drugs in veterinary urine samples. Keywords: Multi-walled carbon nanotubes, solid bar microextraction, non-steroidal anti-inflammatory drugs, urine, liquid chromatography, ketoprofen.

Serological and molecular evidence of coxiellosis and risk factors in sheep flocks in central-eastern Tunisia.

In this study, we conducted an investigation to determine the true prevalence of coxiellosis in sheep in central-eastern Tunisia. A total of 492 veterinary samples taken from 110 flocks were screened for coxiellosis using IS1111-based real-time PCR assay. Sheep sera were tested using an indirect enzyme-linked immunosorbent assay. Based on molecular and serological results, the true adjusted animal and herd-level prevalence of coxiellosis were 11.8% and 20.21%, respectively. Bacterial excretion was observed in 17 flocks, and 19 females showed evidence of Coxiella burnetii shedding (100%). In addition, a statistically significant association was found between vaginal and milk shedding for sheep. Multivariable logistic regression analysis at the animal-population level indicated that strata and vaccination variables were found to be associated with coxiellosis. Besides, it was shown that this infection increased when the intensive farm was exposed to carnivores and when the cleaning practices were not respected, while it decreased when a suitable quarantine was introduced for any introduction of a new animal. Good hygiene and sanitation practices on-farm should be handled as strategies to deal with this zoonotic pathogen in herds.

Kanamycin detection at graphene quantum dot-decorated gold nanoparticles in organized medium after solid-phase extraction using an aminoglycoside imprinted polymer

This is a description of the indirect determination of kanamycin sulfate though the photoluminescence enhancement of an aqueous dispersion of amino-functionalized graphene quantum dots (amino-GQDs) coupled with gold nanoparticles (AuNPs) in a cationic surfactant-rich medium. Specifically, cetyltrimethylammonium bromide (CTAB) was used as the cationic surfactant in our work. Previously, solid phase extraction with a cartridge packed with aminoglycoside-selective imprinted polymer ensured selectivity in kanamycin determination in yellow-fever vaccine and veterinary pharmaceutical samples. The proposed method has trace analysis capability and it is simple to perform as it does not involve the use of toxic reagents employed for chemical derivatization of aminoglycoside antibiotics.

Molecular prevalence of Chlamydia and Chlamydia-like bacteria in Tunisian domestic ruminant farms and their influencing risk factors.

Chlamydia and Chlamydia-like bacteria are well known to infect several organisms and may cause a wide range of diseases, particularly in ruminants. To gain insight into the prevalence and diversity of these intracellular bacteria, we applied a pan-Chlamydiales real-time PCR to 1,134 veterinary samples taken from 130 Tunisian ruminant herds. The true adjusted animal population-level prevalence was 12.9% in cattle, against 8.7% in sheep. In addition, the true adjusted herd-level prevalence of Chlamydiae was 80% in cattle and 25.5% in sheep. Chlamydiales from three family-level lineages were detected indicating a high biodiversity of Chlamydiales in ruminant herds. Our results showed that Parachlamydia acanthamoebae could be responsible for bovine and ovine chlamydiosis in central-eastern Tunisia. Multivariable logistic regression analysis at the animal population level indicated that strata and digestive disorders variables were the important risk factors of bovine and ovine chlamydiosis. However, origin and age variables were found to be associated with bovine and ovine chlamydiosis, respectively. At the herd level, risk factors for Chlamydia positivity were as follows: abortion and herd size for cattle against breeding system, cleaning frequency, quarantine, use of disinfectant and floor type for sheep. Paying attention to these risk factors will help improvement of control programs against this harmful zoonotic disease.

Pseudomonas aeruginosa variants obtained from veterinary clinical samples reveal a role for cyclic di-GMP in biofilm formation and colony morphology.

Overuse of antibiotics is contributing to an emerging antimicrobial resistance crisis. To better understand how bacteria adapt tolerance and resist antibiotic treatment, Pseudomonas aeruginosa isolates obtained from infection sites sampled from companion animals were collected and evaluated for phenotypic differences. Selected pairs of clonal isolates were obtained from individual infection samples and were assessed for antibiotic susceptibility, cyclic di-GMP levels, biofilm production, motility and genetic-relatedness. A total of 18 samples from equine, feline and canine origin were characterized. A sample from canine otitis media produced a phenotypically heterogeneous pair of P. aeruginosa isolates, 42121A and 42121B, which during growth on culture medium respectively exhibited hyper dye-binding small colony morphology and wild-type phenotypes. Antibiotic susceptibility to gentamicin and ciprofloxacin also differed between this pair of clonal isolates. Sequence analysis of gyrA, a gene known to be involved in ciprofloxacin resistance, indicated that 42121A and 42121B both contained mutations that confer ciprofloxacin resistance, but this did not explain the differences in ciprofloxacin resistance that were observed. Cyclic di-GMP levels also varied between this pair of isolates and were shown to contribute to the observed colony morphology variation and ability to form a biofilm. Our results demonstrate the role of cyclic di-GMP in generating the observed morphological phenotypes that are known to contribute to biofilm-mediated antibiotic tolerance. The generation of phenotypic diversity may go unnoticed during standard diagnostic evaluation, which potentially impacts the therapeutic strategy chosen to treat the corresponding infection and may contribute to the spread of antibiotic resistance.

CHROMagar COL-APSE: a selective bacterial culture medium for the isolation and differentiation of colistin-resistant Gram-negative pathogens.

A selective chromogenic culture medium for the laboratory isolation and differentiation of colistin resistant Acinetobacter, Pseudomonas, Stenotrophomonas and Enterobacteriaceae spp. (CHROMagar COL-APSE) was developed, evaluated and compared to an existing selective bacterial culture medium (SuperPolymyxin). The medium was challenged with 84 isolates, including polymyxin B (POL B)-susceptible and -resistant type strains and colistin (COL)-resistant organisms recovered from human and animal samples. Susceptibility to COL and POL B was determined by agar dilution and broth microtitre dilution. The lower limit for the detection of COL-resistant organisms was also calculated for both CHROMagar COL-APSE and SuperPolymyxin media. The ability to isolate and correctly differentiate COL-resistant organisms within mixed cultures was also assessed and compared using both media. Using CHROMagar COL-APSE, Gram-negative pathogens (n=71) with intrinsic (n=8) or acquired COL (n=63) resistance were recovered with 100 % specificity down to the lower limit of detection of 101 colony-forming units (c.f.u.). The growth on SuperPolymyxin was similar, but notably weaker for COL-resistant non-fermentative bacteria (Acinetobacter, Pseudomonas and Stenotrophomonas). CHROMagar COL-APSE was also more sensitive in supporting the growth of Enterobacteriaceae with COL resistance associated with the carriage of mcr-1. CHROMagar COL-APSE is a sensitive and specific medium for the growth of COL-resistant bacterial pathogens. Due to the low limit of detection (101 c.f.u.), it may be useful as a primary isolation medium in the surveillance and recovery of COL-resistant bacteria from complex human, veterinary and environmental samples, especially those with plasmid-mediated MCR-1 or novel mechanisms of polymyxin resistance.

Detection of Salmonella spp. in veterinary samples by combining selective enrichment and real-time PCR.

Rapid screening for enteric bacterial pathogens in clinical environments is essential for biosecurity. Salmonella found in veterinary hospitals, particularly Salmonella enterica serovar Dublin, can pose unique challenges for culture and testing because of its poor growth. Multiple Salmonella serovars including Dublin are emerging threats to public health given increasing prevalence and antimicrobial resistance. We adapted an automated food testing method to veterinary samples and evaluated the performance of the method in a variety of matrices including environmental samples ( n = 81), tissues ( n = 52), feces ( n = 148), and feed ( n = 29). A commercial kit was chosen as the basis for this approach in view of extensive performance characterizations published by multiple independent organizations. A workflow was established for efficiently and accurately testing veterinary matrices and environmental samples by use of real-time PCR after selective enrichment in Rappaport-Vassiliadis soya (RVS) medium. Using this method, the detection limit for S. Dublin improved by 100-fold over subculture on selective agars (eosin-methylene blue, brilliant green, and xylose-lysine-deoxycholate). Overall, the procedure was effective in detecting Salmonella spp. and provided next-day results.

Molecular characterization of antibiotic resistance in cultivable multidrug-resistant bacteria from livestock manure

Diverse antibiotic-resistance genes (ARGs) are frequently reported to have high prevalence in veterinary manure samples due to extensive use of antibiotics in farm animals. However, the characteristics of the distribution and transmission of ARGs among bacteria, especially among different species of multiple antibiotic-resistant bacteria (MARB), have not been well explored. By applying high-throughput sequencing methods, our study uncovered a vast MARB reservoir in livestock manure. The genera Escherichia, Myroides, Acinetobacter, Proteus, Ignatzschineria, Alcaligenes, Providencia and Enterococcus were the predominant cultivable MARB, with compositions of 40.6%–85.7%. From chicken manure isolates, 33 MARB were selected for investigation of the molecular characteristics of antibiotic resistance. A total of 61 ARGs and 18 mobile genetic elements (MGEs) were investigated. We found that 47 ARGs were widely distributed among the 33 MARB isolates. Each isolate carried 27–36 genes responsible for resistance to eight classes of antibiotics frequently used in clinic or veterinary settings. ARGs to the six classes of antibiotics other than streptogramins and vancomycin were present in all 33 MARB isolates with a prevalence of 80%–100%. A total of 12 MGEs were widely distributed among the 33 MARB, with intI1, IS26, ISaba1, and ISEcp1 simultaneously present in 100% of isolates. In addition, 9 gene cassettes within integrons and ISCR1 were detected among MARB isolates encoding resistance to different antibiotic classes. This is the first report revealing the general co-presence of multiple ARGs, various MGEs and ARG cassettes in different species of individual MARB isolates in chicken manure. The results highlight a much higher risk of ARGs spreading through livestock manure to humans than we expected.

Phenotypic and Molecular Characterization of Salmonella 1,4,[5],12:i:- R-Type ASSuT Isolates from Humans, Animals, and Environment in Portugal, 2006-2011.

The increase in prevalence of Salmonella 1,4,[5],12:i:- related infections over the last few years has been considered a public health issue in many European countries, especially as this serovar may be associated with tetraresistance to ampicillin, streptomycin, sulfonamides, and tetracyclines (R-type ASSuT). Salmonella 1,4,[5],12:i:- isolates (n = 187) obtained by the Portuguese National Laboratory from different sources, including human clinical cases (n = 170), veterinary (n = 10), environmental (n = 6), and food samples (n = 1), were collected from 15 districts between 2006 and 2011. All isolates were serotyped using the slide agglutination method and results were confirmed by multiplex PCR for the monophasic variant. From the confirmed Salmonella 1,4,[5],12:i:-, R-type ASSuT isolates were selected by disc diffusion and minimal inhibitory concentration (MIC) determination for further characterization by pulsed-field gel electrophoresis restriction with XbaI, virulence genes determination by PCR, additional antimicrobial resistance profiling by disc diffusion, and epidemiological distribution evaluation. Out of the 187 serotyped isolates, 133 were confirmed as Salmonella 1,4,[5],12:i:- with a R-type ASSuT occurrence of 61.7%. Distribution among Portuguese districts showed a higher percentage of reported cases in coastal areas, in particular, in Porto (24.8%), Setúbal (13.5%), and Aveiro (12.8%), probably due to the higher population density. Clonality analysis revealed a high diversity of pulsotypes with the majority of human salmonellosis cases being attributed to sporadic events. All isolates harbored 14 out of the 18 virulence genes evaluated and 87.8% of the isolates showed all the resistance genes frequently associated with the European clone, blaTEM+sul2+straA-straB+tetB+. This study shows that Salmonella 1,4,[5],12:i:- resistant isolates are widely distributed in Portugal. This may be related to a selective advantage offered by R-type ASSuT profile, the presence of multiple virulent features, including the ability to form biofilms, which along with a high diversity of pulsotypes may be responsible for the dissemination through the country.

Determination of N-acylhomoserine lactones of Pseudomonas aeruginosa in clinical samples from dogs with otitis externa.

BackgroundBacterial intercellular communication, called quorum sensing, takes place via the production and collective response to signal molecules. In Gram-negative bacteria, like Pseudomonas aeruginosa, these signaling molecules are N-acylhomoserine lactones (AHLs). P. aeruginosa is a common cause of inflammation of the ear canal (otitis externa) in dogs. It employs quorum sensing to coordinate the expression of host tissue-damaging factors, which are largely responsible for its virulence. The treatment of P. aeruginosa-associated otitis is challenging due to a high intrinsic resistance of P. aeruginosa to several antibiotics.Attenuation of quorum sensing signals to inhibit bacterial virulence is a novel strategy for the treatment of resistant bacterial pathogens, including P. aeruginosa. Therefore, it is important to recognize and define quorum sensing signal molecules in clinical samples. To date, there are no reports on determination of AHLs in the veterinary clinical samples. The purpose of this study was to validate an analytical procedure for determination of the concentration of AHLs in the ear rinses from dogs with P. aeruginosa-associated otitis externa.Samples were obtained with rinsing the ear canals with physiological saline solution. For validation, samples from healthy dogs were spiked with none or different known amounts of the selected AHLs. With the validated procedure, AHLs were analyzed in the samples taken in weekly intervals from two dogs, receiving a standard treatment for P. aeruginosa-associated otitis externa.ResultsValidation proved that the procedure enables quantification of AHLs in non-clinical and clinical samples. In addition, a time dependent reduction of AHL concentration was detected for the treated dogs.ConclusionsOur results indicate that liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) is superior in detecting AHLs compared to other chromatographic techniques. This is the first report on determination of AHLs in the clinical samples of veterinary importance. The analytical procedure described in this paper is capable of supporting novel antimicrobial strategies, which target quorum sensing.

A mixed methods study of ruminant brucellosis in central-eastern Tunisia.

In this study, we conducted an investigation to determine the true prevalence of bovine and ovine brucellosis in central-eastern Tunisia. A total of 1134 veterinary samples taken from 130 ruminant herds were screened for brucellosis using IS711-based real-time PCR assay. Sera collected from the ruminants were tested using the Rose Bengal test and indirect enzyme-linked immunosorbent assay. Based on serological and molecular results, the true adjusted animal population level prevalence was 23.5% in cattle, against 13.5% in sheep. In addition, the true adjusted herd level prevalence of brucellosis was 55.6% in cattle and 21.8% in sheep. A statistically significant association was found between vaginal and milk shedding for ruminants. In addition, our results showed that Brucella abortus could be responsible for bovine and ovine brucellosis. Multivariable logistic regression analysis at the animal population level indicated that age and origin variables were important risk factors for cattle. However, age and abortion variables were found to be associated with ovine brucellosis. At the herd level, risk factors for Brucella positivity were as follows: abortion and herd composition for cattle against herd composition, mortality rates, and hygiene for sheep. Animal hygiene, food quality, and sanitary practices on the farm should be applied as strategies to control brucellosis in herds.

Detection of wide genetic diversity and several novel strains among non-avium nontuberculous mycobacteria isolated from farmed and wild animals in Hungary.

Besides Mycobacterium avium numerous nontuberculous Mycobacterium (NTM) species exist, which pose constant health risk to both humans and animals. The aim of our study was to identify non-avium NTM isolates from veterinary origin in Hungary, and to detect the occurrence of rifampicin resistance among them. Two hundred and twenty-five strains isolated between 2006 and 2013 from domestic and wild animals and veterinary important samples were identified on the basis of partial DNA sequences of different structural or coding genes, besides commercial kits and multiplex PCR. From 14 different sources, 28 NTM strains and 8 hitherto unidentified strain types were detected. Mycobacterium nonchromogenicum was the most frequently occurring strain (25·78%). Besides, new hosts and mycobacteria-related pathological symptoms were detected. Noticeable rifampicin resistance (42·76%) was found among 159 strains from six different host species. Furthermore, we described the problematics of strain-misidentifications using commercial kits. Our study identified the most common non-avium NTM strains in Hungary, and provided account of their occurrence, host range, and pathogenicity. The detected high rifampicin resistance among the strains isolated mainly from fallow and red deer clearly shows that more attention should be paid to the examination of wild animals especially to those ones which may have contact or shared territory with farmed animals. In domestic animal husbandry the maintenance of tuberculosis free status is of primary importance. As immunological cross-reactions due to NTM hamper the diagnosis of bovine tuberculosis, the precise identification of NTM strains would be essential in the veterinary diagnostics, especially for potentially zoonotic strains. This is the first study investigating the strain diversity of non-avium NTM in Hungary.

Simultaneous detection of Waddlia chondrophila and Listeria monocytogenes in aborted ruminant samples by real-time quantitative PCR

Waddlia chondrophila and Listeria monocytogenes are well known emerging pathogens that cause ruminants' abortion around the world. Zoonotic infections caused by these bacteria are mostly underestimated due to difficulties of diagnosis resulting from their intracellular growth. The purpose of this study was to develop and validate a dual real-time quantitative PCR (qPCR) assay for the simultaneous quantification of W. chondrophila and L. monocytogenes in biological samples from aborted ruminants. Technical performance was examined using linear control plasmids. A total of 211 veterinary samples (15 placental tissues, 50 blood, 53 milk and 93 vaginal swab samples) were used to compare the qPCR with standard culture methods. The limit of detection was 2 plasmid template copies per reaction with approximately 5000-fold differences in concentrations of the two competing templates. Coefficients of variation for positive control plasmids were less than 1.8%. Both intra- (1.01–2.13% and 1.13–1.71%) and inter- (1.02–2.24% and 1.2–1.66%) assay variations of qPCR for W. chondrophila and L. monocytogenes plasmids were within the acceptable limits, implying high reproducibility and repeatability of the assay. The use of the qPCR assay resulted in 53 positive identifications among 211 veterinary samples against only 19 when standard cultural methods were used. On the basis of these results, we determined 100% sensitivity and 100% specificity for our new qPCR. Dual abortigenic agents were identified in 8.6% and 26.6% of vaginal swab samples and placental tissues, respectively. To conclude, this new qPCR will be of great value in the simultaneous and rapid diagnosis of W. chondrophila and L. monocytogenes in large-scale screening programs and also during outbreaks.

Review on Diagnostic Cytology: Techniques and Applications in Veterinary Medicine

Cytology is a science of cells that is used to differentiate between normal cells, neoplastic cells and inflamed cells. It has great acceptance than any other diagnostic methods due to its quickness, inexpensiveness and simplicity. It uses the techniques such as fine needle aspiration, impression, scraping, swabs, centesis and catheterization for sample collection. Also slide preparation uses a simple technique such as squash, blood smear; needle spread and line smear techniques. What makes cytology unique is that it can tell us the result of the diseases while the patient is with us. This science has also wide application on determining the external and internal diseases including neoplastic diseases. In case of neoplastic diseases it is important to differentiate between neoplastic nucleus, neoplastic cytoplasm and neoplastic structure of different cells with their normal cells. In addition to these, it also used in direct therapy, to form prognosis and to determinate next diagnostic procedures. Character of the lesion and tissue sampled play pivotal roles in the diagnostic value of cytology. Familiarity with preferred sampling methods and reported accuracy is critical for veterinary practitioners and sample of good quality is imperative. Different specimen sampling methods and specimen processing methods should be properly practiced.

The impact of biosecurity and partial depopulation on Campylobacter prevalence in Irish broiler flocks with differing levels of hygiene and economic performance

BackgroundCampylobacter jejuni is the leading bacterial food-borne pathogen within the European Union (EU), and poultry meat is the primary route for transmission to humans.Material and methodsThis study examined the impact of partial depopulation (thinning), season, and farm performance (economic, hygiene, and biosecurity) on Campylobacter prevalence in Irish broilers over a 13-month period. Ten caecal samples were taken per flock, for a total of 211 flocks from 23 farms during the duration of the study. Campylobacter was isolated and enumerated according to modified published ISO methods for veterinary samples. Biosecurity was evaluated through a questionnaire based on risk factors for Campylobacter identified in previous studies. Hygiene compliance was assessed from audit records taken over the course of 1 year. All information relating to biosecurity and hygiene was obtained directly from the processing company. This was done to ensure farmers were unaware they were being monitored for Campylobacter prevalence and prevent changes to their behaviour.Results and discussionFarms with high performance were found to have significantly lower Campylobacter prevalence at first depopulation compared with low-performance farms across all seasons (P≤0.01). Peak Campylobacter levels were observed during the summer season at first thin in both the high- and low-performance groups. Campylobacter prevalence was found to increase to ≥85% in both high- and low-performance farms across all seasons at final depopulation, suggesting that Campylobacter was introduced during the first depopulation. On low-performance farms, four biosecurity interventions were found to significantly reduce the odds of a flock being Campylobacter positive (physical step-over barrier OR=0.17, house-specific footwear OR=0.13, absence of water body within 0.5 km OR=0.13, two or more broiler houses on a farm OR=0.16), compared with farms without these interventions. For high-performance farms, no single biosecurity intervention was identified as significant as this group had full compliance with multiple factors. High-performance farms had significantly better feed conversion ratios compared with low-performance farms (1.61 v 1.67 (P≤0.01)). No differences in flock mortality rates were observed (P≥0.05). This highlights the impact of season, biosecurity, partial depopulation, and farm performance on Campylobacter prevalence in Irish broilers.