Veterinary Reference Laboratories Research Articles

Comparative study of a broad qPCR panel and centrifugal flotation for detection of gastrointestinal parasites in fecal samples from dogs and cats in the United States

BackgroundFor decades, zinc sulfate centrifugal fecal flotation microscopy (ZCF) has been the mainstay technique for gastrointestinal (GI) parasite screening at veterinary clinics and laboratories. Elsewhere, PCR has replaced microscopy because of generally increased sensitivity and detection capabilities; however, until recently it has been unavailable commercially. Therefore, the primary aim of this study was to compare the performance of real-time PCR (qPCR) and ZCF for fecal parasite screening. Secondary aims included further characterization of markers for hookworm treatment resistance and Giardia spp. assemblages with zoonotic potential and qPCR optimization.MethodsA convenience sampling of 931 canine/feline fecal samples submitted to a veterinary reference laboratory for routine ZCF from the Northeast US (11/2022) was subsequently evaluated by a broad qPCR panel following retention release. Detection frequency and agreement (kappa statistics) were evaluated between ZCF and qPCR for seven GI parasites [hookworm/(Ancylostoma spp.), roundworm/(Toxocara spp.), whipworm/(Trichuris spp.), Giardiaduodenalis, Cystoisospora spp., Toxoplasmagondii, and Tritrichomonasblagburni] and detections per sample. Total detection frequencies were compared using a paired t-test; positive sample and co-infection frequencies were compared using Pearson’s chi-squared test (p ≤ 0.05 significant) and qPCR frequency for hookworm benzimidazole (BZ) resistance (F167Y) and zoonotic Giardia spp. assemblage markers calculated. Confirmatory testing, characterization, and qPCR optimization were carried out with Sanger sequencing.ResultsqPCR detected a significantly higher overall parasite frequency (n = 679) compared to ZCF (n = 437) [p = < 0.0001, t = 14.38, degrees-of-freedom (df) = 930] and 2.6 × the co-infections [qPCR (n = 172) vs. ZCF (n = 66)], which was also significant (p = < 0.0001, X2 = 279.49; df = 1). While overall agreement of parasite detection was substantial [kappa = 0.74; (0.69–0.78], ZCF-undetected parasites reduced agreement for individual and co-infected samples. qPCR detected markers for Ancylostomacaninum BZ resistance (n = 5, 16.1%) and Giardia with zoonotic potential (n = 22, 9.1%) as well as two parasites undetected by ZCF (T.gondii/T.blagburni). Sanger sequencing detected novel roundworm species, and qPCR optimization provided detection beyond ZCF.ConclusionsThese results demonstrate the statistically significant detection frequency advantage offered by qPCR compared to routine ZCF for both single and co-infections. While overall agreement was excellent, this rapid, commercially available qPCR panel offers benefits beyond ZCF with detection of markers for Giardia assemblages with zoonotic potential and hookworm (A.caninum) BZ resistance.Graphical

European Union Reference Laboratories support the National food, feed and veterinary Reference Laboratories with rolling out whole genome sequencing in Europe.

The Inter European Union Reference Laboratories (EURLs) Working Group on Next Generation Sequencing (NGS) involves eight EURLs for microbiological food and feed hazards and has been working since 2017 to promote the adoption of NGS by the National Reference Laboratories (NRLs) in the European Union. This work illustrates the results of the first 5 years of activity. By working together, the EURLs involved have released guidance documents for assisting NRLs in all the steps of NGS, helping the transition from classical molecular methods towards whole genome sequencing while ensuring harmonization, with the final aim of improving preparedness in the use of NGS to characterize microbial hazards and trace the sources of infection.

Comparison of Biochemical Test Results with a Portable Point-of-Care Analyzer and a Reference Laboratory Analyzer in Eastern Indigo Snakes (Drymarchon couperi)

Eastern indigo snakes (EIS), Drymarchon couperi, have unique physiological features that are not well understood. A useful tool to better understand the physiology of this species are reference intervals for plasma biochemical analytes. However, reported values for plasma biochemical analytes are often specific to the analyzer used. In order to determine whether a point of care analyzer would produce results comparable to a veterinary reference laboratory analyzer, this study aimed to compare plasma biochemical analyte values between a point of care analyzer and a veterinary reference lab analyzer, regardless of EIS health status. Plasma biochemical analyte values were obtained from 31 EIS and compared between analyzers. The analytes compared were albumin (ALB), aspartate aminotransferase (AST), total calcium (Ca), creatinine kinase (CK), glucose (GLU), potassium (K), sodium (Na), phosphorus (P), total protein (TP), and uric acid (UA). Analytes with a very high correlation between analyzers were AST (r = 0.923), K (r = 0.916), and UA (r = 0.965); analytes with a high correlation were Ca (r = 0.724), CK (r = 0.789), GLU (r = 0.889), and TP (r = 0.812); moderate correlation P (r = 0.528); low correlation ALB (r = 0.471); and no correlation Na (r = -0.138). A point of care analyzer offers many benefits such as portability, rapid results, small sample size requirements, and the ability to use whole blood. The Vetscan VS2 produces similar values for only some analytes signifying the need for method-specific reference intervals for plasma biochemical analytes in EIS.

Microbiological Diagnoses on Clinical Mastitis-Comparison between Diagnoses Made in Veterinary Clinics versus in Laboratory Applying MALDI-TOF MS.

The present study compares the diagnoses on clinical bovine mastitis made in veterinary clinics using conventional diagnostic methods with diagnoses on the same samples made by a veterinary reference laboratory using MALDI-TOF MS as diagnostics. The study enables targeted and evidence-based consulting on prudent mastitis diagnostics and related antibiotic usage. In total, 492 samples from clinical mastitis were included. When applying MALDI-TOF MS as gold standard, only 90 out of 492 diagnoses made in veterinary clinics, equal to 18%, were correct. Four main findings were important: (1) the veterinary clinics overlooked contamination in mastitis samples; (2) the veterinary clinics only assigned 2 fully correct diagnoses out of 119 samples with mixed growth cultures; (3) the veterinary clinics made close to half of their diagnoses on pure culture erroneously; (4) the veterinary clinics applied a limited number of the relevant pathogen identifications on pure culture samples. Altogether, the present study shows that a large part of Danish clinical mastitis cases are misdiagnosed. Lack of correct diagnoses and diagnostic quality control may lead to the choice of wrong treatment and thus hamper prudent use of antibiotics. Hence, the present study warns a risk of overuse of antibiotics in Denmark. Consequently, the present study calls for training of veterinary clinics in diagnostics of mastitis pathogens and national guidelines on quality assurance of mastitis diagnostics.

Salmonella Typhimurium in Iran: Contribution of molecular and IS200 PCR methods in variants detection.

Salmonella Typhimurium, a zoonotic pathogen, is regarded as a major health and economic concern worldwide. Recently, monophasic variants of this serovar have been significantly associated with human gastroenteritis outbreaks globally, making its accurate identification essential for epidemiological and control purposes. We have identified and analyzed 150 S. Typhimurium from 884 Salmonella genus isolated from humans, domestic animals, poultry, food items and abattoirs origins. The Salmonella isolates were obtained from Iranian National Veterinary Reference Laboratories of 9 provinces during 2007–2016, and from five hospitals in Tehran in 2015. The isolates were evaluated biochemically, serologically, and by PCR amplification of invA, mdh, STM4492, fliC, fljA, fljB, hin genes, IS200 and DT104. invA and mdh genes were used to confirm the S. Typhimurium serotype, fliC and fljB genes for determination of monophasic variants and amplification of IS200 to discriminate the monophasic variants from the closely related serotypes. We identified 78.6% (118/150) as classical S. Typhimurium (fliC, fljB and IS200 positive), 12.6% (19/150) were IS200 negative from all isolates. DT104 is another marker for S.Typhimurium serovar typing. Contrary to EFSA guidelines 20.6% (19/29) of human isolates that lacked IS200 insertion sequence, were confirmed as S.Typhimurium. Compared to the North American/European isolates the low prevalence of fljB negative 6% (9/150) and the high abundance of fliC negative 23.3% (35/150) isolates also were indicative of a different regional atypical population. Studies have shown that the prevalence of monophasic (fljB-) S. Typhimurium worldwide is promoted by the Swine industry. Thus, one reason for this high number of different atypical strains could be inhibition of swine breeding system (house hold and industry) in Iran. These results demonstrate a need for a modified identifying protocol to overcome the regional differences.

Twenty years of Hendra virus: laboratory submission trends and risk factors for infection in horses.

Hendra virus (HeV) was first described in 1994 in an outbreak of acute and highly lethal disease in horses and humans in Australia. Equine cases continue to be diagnosed periodically, yet the predisposing factors for infection remain unclear. We undertook an analysis of equine submissions tested for HeV by the Queensland government veterinary reference laboratory over a 20-year period to identify and investigate any patterns. We found a marked increase in testing from July 2008, primarily reflecting a broadening of the HeV clinical case definition. Peaks in submissions for testing, and visitations to the Government HeV website, were associated with reported equine incidents. Significantly differing between-year HeV detection rates in north and south Queensland suggest a fundamental difference in risk exposure between the two regions. The statistical association between HeV detection and stockhorse type may suggest that husbandry is a more important risk determinant than breed per se. The detection of HeV in horses with neither neurological nor respiratory signs poses a risk management challenge for attending veterinarians and laboratory staff, reinforcing animal health authority recommendations that appropriate risk management strategies be employed for all sick horses, and by anyone handling sick horses or associated biological samples.

Effects of Ultracentrifugation on Plasma Biochemical Values of Prefledged Wild Peregrine Falcons (Falco peregrinus) in Northeastern Illinois

Centrifugation is performed on whole blood samples to obtain serum or plasma for biochemical analysis. Although blood samples centrifuged in a microhematocrit tube may maximize recovery of plasma from small-volume samples, plasma biochemical values from such samples have been implicated as causing erroneous results. To compare blood biochemical values obtained by microhematocrit centrifugation and centrifugation with a commercial tilt-rotor machine, blood samples were collected from peregrine falcon (Falco peregrinus) eyases aged 32-40 days (n=51). The samples were separated into 2 equal aliquots with 1 aliquot centrifuged in a tilt-rotor machine and the other aliquot ultracentrifuged in microhematocrit tubes. Separated plasma from both processes was sent to a commercial veterinary reference laboratory for routine clinical biochemical analysis. No significant differences were found in the biochemical results of the paired samples by the 2 centrifugation methods. These results show that the centrifugation method has no effect on the plasma quality for biochemical analysis in young peregrine falcons.

Evaluation of Coccidioides Antigen Detection in Dogs with Coccidioidomycosis

Antigen detection has been reported to be a promising method for rapid diagnosis of coccidioidomycosis in humans. Coccidioides antigen detection has not been previously reported in dogs with coccidioidomycosis and was evaluated in 60 cases diagnosed based on detection of anti-Coccidioides antibodies at titers of 1:16 or more in serum. Controls included dogs with presumed histoplasmosis or blastomycosis, other fungal infections, or nonfungal diseases and healthy dogs. Urine and serum specimens were tested using an enzyme immunoassay for Coccidioides galactomannan antigen. Antibody testing was performed at commercial veterinary reference laboratories. Antigen was detected in urine or serum of 12 of 60 (20.0%), urine only in 2 of 57 (3.5%), and serum only in 11 of 58 (19.0%) dogs with coccidioidomycosis. Antigen was detected in the urine of 3 of 43 (7.0%) and serum of 1 of 37 (2.7%) dogs with histoplasmosis or blastomycosis but not in 13 dogs with other fungal infections (serum, 9; urine, 13), 41 dogs with nonfungal diseases (urine, 41; serum, 18), or healthy dogs (serum, 21; urine, 21). Detection of antigen was an insensitive method for diagnosis of coccidioidomycosis in dogs in which the diagnosis was based primarily upon detection of antibodies at titers of 1:16 or higher, and the highest sensitivity was in serum.

Vitamin D-dependent non-type 1, non-type 2 rickets in a 3-month-old Cornish Rex kitten

CASE PRESENTATION AND ASSESSMENT: A 3-month-old female Cornish Rex kitten was found to have non-painful swelling of the carpal and tarsal regions when presented for routine neutering. The kitten was smaller in stature and less active than its siblings and, according to the owner, had a bunny-hopping gait, was reluctant to climb stairs and strained during defecation. Radiography of the affected limbs and a subsequent radiographic survey of the entire skeleton demonstrated features consistent with rickets. The three littermates were clinically and radiographically normal. As a nutritionally complete diet was being fed, it seemed most likely that the kitten had an inborn error related to vitamin D metabolism. Serum biochemistry demonstrated reduced total alkaline phosphatase activity and increased concentrations of parathyroid hormone. Concentrations of 1,25- and 25-hydroxycholecalciferol were markedly reduced, confirming the diagnosis of rickets. The kitten was treated with calcitriol, administered orally once daily, and improved rapidly both clinically and radiologically. Serial laboratory studies suggested that the error in vitamin D metabolism was transient, and, at the time of writing, as an adult, the cat appears to require no ongoing replacement calcitriol therapy. This case emphasises the value of examining a full 'calcium profile' via a human or veterinary reference laboratory, and a favourable prognosis in some kittens with rickets makes such investigations worthwhile. Even when finances preclude detailed investigation, trial therapy using a nutritionally complete diet and physiological doses of calcitriol or cholecalciferol is inexpensive and can produce a good response.

Aufgaben der veterinärmedizinischen Referenzlaboratorien im Bereich der lebensmittelbedingten Zoonosen

Reference laboratories are of central importance for consumer protection. Field expertise and high scientific competence are basic requirements for the nomination of a national reference laboratory. To ensure a common approach in the analysis of zoonotic hazards, standards have been developed by the reference laboratories together with national official laboratories on the basis of Art. 33 of Directive (EG) No. 882/2004. Reference laboratories function as arbitrative boards in the case of ambivalent or debatable results. New methods for detection of zoonotic agents are developed and validated to provide tools for analysis, e. g., in legal cases, if results from different parties are disputed. Besides these tasks, national reference laboratories offer capacity building and advanced training courses and control the performance of ring trials to ensure consistency in the quality of analyses in official laboratories. All reference laboratories work according to the ISO standard 17025 which defines the grounds for strict laboratory quality rules and in cooperation with the respective Community Reference Laboratories (CRL). From the group of veterinary reference laboratories for food-borne zoonoses, the national reference laboratories are responsible for Listeria monocytogenes, for Campylobacter, for the surveillance and control of viral and bacterial contamination of bivalve molluscs, for E. coli, for the performance of analysis and tests on zoonoses (Salmonella), and from the group of parasitological zoonotic agents, the national reference laboratory for Trichinella.

Standardized Pulsed-Field Gel Electrophoresis of Shiga Toxin–Producing Escherichia coli: The PulseNet Europe Feasibility Study

PulseNet USA, the American molecular subtyping network for foodborne infections, has since 1996 been highly successful combating infections caused by Shiga toxin-producing Escherichia coli (STEC) O157, Salmonella, Listeria, and Shigella spp. The PulseNet Europe feasibility study was initiated to ascertain the interest of public health and veterinary reference laboratories to establish a similar network, and to determine if it was possible to perform standardized pulsed-field gel electrophoresis (PFGE) typing of Salmonella, Listeria, and STEC on a large scale in Europe. The results of the STEC part of that study are presented here. Twenty-seven veterinary and public health laboratories participated in the study. The participants subtyped eight E. coli strains by PFGE using the restriction enzyme XbaI according to the PulseNet or a similar protocol, with strict adherence to the electrophoretic conditions stated in the former and submitted an image of their gel for centralized anonymous analysis. The quality of the gels was first graded visually as "good," "intermediate," and "unsatisfactory." The number of gels graded this way was 11, 14, and 2, respectively. All "good" and "intermediate" gels were also analysed and compared by computerized analysis to a reference gel. For gels graded "good," on average 5.6, 7.4, and 8 patterns out of 8 per gel were identified with a similarity of 100%, >95%, and >90%, respectively. The corresponding numbers for gels graded "intermediate" were 1.7, 4.9, and 7.4, respectively. The problems causing the grading to be "intermediate" was overloaded lanes, overexposed images, not optimally focused images and incomplete digestion, all problems that led to misinterpretation of the number of restriction fragments in the gel. These problems may be corrected by simple adjustments to the subtyping procedure. Thus, there seems to be little need for training of the participants in PulseNet Europe.

Resistance of Salmonella Isolates in Germany

During 2000-2002 the National Veterinary Reference Laboratory for Salmonella (NRL-Salm) in Germany typed 11,911 isolates from animals, food, feed and the environment. All of them were tested for their susceptibility to 17 anti-microbial agents. Sixty-three per cent of all isolates were resistant and 40% were multiresistant (resistant against more than one anti-microbial). This general resistance level was strongly influenced by those specific serotypes which dominate the Salmonella epidemiology in Germany. Salmonella Typhimurium DT104 isolates from pig and cattle, and their resulting food products, were multiresistant in 98 and 94% of the cases respectively. During the period 2000-2003 an increasing quinolone resistance especially in Salmonella isolates from poultry and poultry meat (to 26%) and in S. Paratyphi B D-tartrate positive isolates (to 64%) could be observed. This increase was accompanied by a shift towards higher minimal inhibitory concentrations for ciprofloxacin.

Reference Values in Veterinary Medicine

In veterinary medicine, as in human medicine, reference values are necessary for useful interpretation of laboratory results. Without appropriate reference values, the sensitivity and specificity of tests are lowered, which can result in erroneous diagnoses and treatment protocols that may cause harm to or death of the animal tested. Reference values specific for the species tested and the equipment and reagents used are essential for accurate interpretation. Many veterinary reference laboratories use historic or published values rather than establishing their own, because of the time and expense involved. In addition, limited availability of sufficient numbers of many species (40–120 or more animals) makes establishment of laboratory-specific reference values impractical.

National and international veterinary reference laboratories for infectious diseases.

Reference laboratories play an increasingly important role in the harmonisation of laboratory diagnostic tests and the standardisation of veterinary vaccines. This is particularly important in building confidence between international trading partners. The authors review aspects of the organisation, designation and support of reference laboratories for infectious diseases of animals and discuss the principal activities which such laboratories would normally perform. These activities include advice and consultancy, publications and communication, training, research, disease surveillance, maintenance of culture collections, evaluation of reference methods, preparation of reference materials and organisation of inter-laboratory comparisons.