Disturbed spermatogenic signaling in pituitary adenylate cyclase activating polypeptide-deficient mice.

PACAP is a neuropeptide with diverse functions in various organs, including reproductive system. It is present in the testis in high concentrations, and in addition to the stage-specific expression within the seminiferous tubules, PACAP affects spermatogenesis and the functions of Leydig and Sertoli cells. Mice lacking endogenous PACAP show reduced fertility, but the possibility of abnormalities in spermatogenic signaling has not yet been investigated. Therefore, we performed a detailed morphological analysis of spermatozoa, sperm motility and investigated signaling pathways that play a role during spermatogenesis in knockout mice. No significant alterations were found in testicular morphology or motility of sperm in homozygous and heterozygous PACAP-deficient mice in spite of the moderately increased number of severely damaged sperms. However, we found robust changes in mRNA and/or protein expression of several factors that play an important role in spermatogenesis. Protein kinase A expression was markedly reduced, while downstream phospho-ERK and p38 were elevated in knockout animals. Expression of major transcription factors, such as Sox9 and phospho-Sox9, was decreased, while that of Sox10, as a redundant factor, was increased in PACAP-deficient mice. The reduced phospho-Sox9 expression was partly due to increased expression and activity of phosphatase PP2A in knockout mice. Targets of Sox transcription factors, such as collagen type IV, were reduced in knockout mice. In summary, our results show that lack of PACAP leads to disturbed signaling in spermatogenesis, which could be a factor responsible for reduced fertility in PACAP knockout mice, and further support the role of PACAP in reproduction.

Altered Notch Signaling in Developing Molar Teeth of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP)-Deficient Mice.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with neuroprotective and neurotrophic effects. This suggests its influence on the development of teeth, which are, similarly to the nervous system, ectoderm and neural crest derivatives. Our earlier studies have shown morphological differences between wild-type (WT) and PACAP-deficient mice, with upregulated sonic hedgehog (SHH) signaling in the lack of PACAP. Notch signaling is a key element of proper tooth development by regulating apoptosis and cell proliferation. In this study, our main goal was to evaluate the possible effects of PACAP on Notch signaling pathway. Immunohistochemical staining was performed of Notch receptors (Notch1, 2, 3, 4), their ligands [delta-like protein (DLL)1, 3, 4, Jagged1, 2], and intracellular target molecules [CSL (CBF1 humans/Su (H) Drosophila/LAG1 Caenorhabditis elegans transcription factor); TACE (TNF-α converting enzyme), NUMB] in molar teeth of 5-day-old WT, and homozygous and heterozygous PACAP-deficient mice. We measured immunopositivity in the enamel-producing ameloblasts and dentin-producing odontoblasts. Notch2 receptor and DLL1 expression were elevated in ameloblasts of PACAP-deficient mice compared to those in WT ones. The expression of CSL showed similar results both in the ameloblasts and odontoblasts. Jagged1 ligand expression was elevated in the odontoblasts of homozygous PACAP-deficient mice compared to WT mice. Other Notch pathway elements did not show significant differences between the genotype groups. The lack of PACAP leads to upregulation of Notch pathway elements in the odontoblast and ameloblast cells. The underlying molecular mechanisms are yet to be elucidated; however, we propose SHH-dependent and independent processes. We hypothesize that this compensatory upregulation of Notch signaling by the lack of PACAP could represent a salvage pathway in PACAP-deficient animals.

Resurgence of rabies in Hungary during 2013-2014: An attempt to track the origin of identified strains.

In 2013-2014, accumulation of rabies episodes raised concerns regarding ongoing elimination programme in Hungary. Nearly four dozen cases were identified over a 13-month period in the central region of the country far behind the immunization zones. Although the outbreak was successfully controlled, the origin of disease remained unknown. In this study, we sequenced the partial N and G genes from 47 Hungarian rabies virus (RV) strains isolated from the 2013-2014 outbreak. Sequencing and phylogenetic analysis of the N and G genes showed that the Hungarian RV isolates share high nucleotide similarity among each other (up to 100%). When analysing the N gene, comparable sequence similarity was seen between the outbreak strains and some historic Romanian RV strains. Unfortunately, in the lack of available sequence data from the Romanian RV strains, the genetic relationship within the G gene could not be determined. Phylogenetic analysis of Hungarian RV isolates detected in the past revealed that multiple independent RV lineages circulated in our country over the past 25 years. The parental strain of the 2013-2014 outbreak may have been imported independently perhaps from east through transborder movement of a reservoir animal. Next to the introduction, this imported RV strain seems to have spread clonally in the affected area. Our findings indicate that despite effective control measures that, overall, minimized the incidence of rabies over the past decade, field and laboratory monitoring needs to be continued to make rabies elimination programme in Hungary successful.

PACAP deficiency as a model of aging.

Dysregulation of neuropeptides may play an important role in aging-induced impairments. In the long list of neuropeptides, pituitary adenylate cyclase-activating polypeptide (PACAP) represents a highly effective cytoprotective peptide that provides an endogenous control against a variety of tissue-damaging stimuli. PACAP has neuro- and general cytoprotective effects due to anti-apoptotic, anti-inflammatory, and antioxidant actions. As PACAP is also a part of the endogenous protective machinery, it can be hypothesized that the decreased protective effects in lack of endogenous PACAP would accelerate age-related degeneration and PACAP knockout mice would display age-related degenerative signs earlier. Recent results support this hypothesis showing that PACAP deficiency mimics aspects of age-related pathophysiological changes including increased neuronal vulnerability and systemic degeneration accompanied by increased apoptosis, oxidative stress, and inflammation. Decrease in PACAP expression has been shown in different species from invertebrates to humans. PACAP-deficient mice display numerous pathological alterations mimicking early aging, such as retinal changes, corneal keratinization and blurring, and systemic amyloidosis. In the present review, we summarize these findings and propose that PACAP deficiency could be a good model of premature aging.

Biomechanical model of corneal transplantation.

PURPOSE: Refractive consequences of corneal transplants are analyzed using corneal biomechanical models assuming homogeneous and inhomogeneous stiffness distributions across the cornea. Additionally, refractive effects of grafts combined with volume removal procedures are also evaluated to develop methods to reduce postoperative refractive management of patients. METHODS: Refinements of a two-dimensional finite element model are applied to simulate the biomechanical and refractive effects of different corneal transplant procedures: anterior lamellar keratoplasty, posterior lamellar keratoplasty, and penetrating keratoplasty. The models are based on a nonlinearly elastic, isotropic formulation. Predictions are compared with published clinical data. RESULTS: The model simulating the penetrating keratoplasty procedure predicts more change in the postoperative corneal curvature than models simulating anterior lamellar keratoplasty or posterior lamellar keratoplasty procedures. When a lenticle-shaped tissue with a central thickness of 50 microns and a diameter of 4 mm is removed from the anterior corneal surface along with the anterior lamellar keratoplasty or posterior lamellar keratoplasty, the models predict a refractive correction of -8.6 and -8.9 diopters, respectively. CONCLUSIONS: Simulations indicate that a posterior lamellar keratoplasty procedure is preferable for obtaining a better corneal curvature profile, eliminating the need for specific secondary treatments.

Finite element analysis applied to cornea reshaping.

A 2-D finite element model of the cornea is developed to simulate corneal reshaping and the resulting deformation induced by refractive surgery. In the numerical simulations, linear and nonlinear elastic models are applied when stiffness inhomogeneities varying with depth are considered. Multiple simulations are created that employ different geometric configurations for the removal of the corneal tissue. Side-by-side comparisons of the different constitutive laws are also performed. To facilitate the comparison, the material property constants are identified from the same experimental data, which are obtained from mechanical tests on corneal strips and membrane inflation experiments. We then validate the resulting models by comparing computed refractive power changes with clinical results. Tissue deformations created by simulated corneal tissue removal using finite elements are consistent with clinically observed postsurgical results. The model developed provides a much more predictable refractive outcome when the stiffness inhomogeneities of the cornea and nonlinearities of the deformations are included in the simulations. Finite element analysis is a useful tool for modeling surgical effects on the cornea and developing a better understanding of the biomechanics of the cornea. The creation of patient-specific simulations would allow surgical outcomes to be predicted based on individualized finite element models.

Myopic intrastromal photorefractive keratectomy with the neodymium-yttrium lithium fluoride picosecond laser in the cat cornea.

OBJECTIVE: To evaluate the refractive effects of myopic intrastromal photorefractive keratectomy (IPRK) with the neodymium-yttrium lithium fluoride (Nd-YLF) picosecond laser. METHODS: A 1053-nm Nd-YLF picosecond laser was used to produce myopic IPRK in cat corneas at threshold energy densities. Preoperative corneal topography and ultrasonic pachymetry were performed followed by IPRK in one eye each of 21 cats. No anti-inflammatory medications were administered. Postoperative corneal topography, ultrasonic pachymetry, and slit-lamp examination were performed at scheduled intervals. RESULTS: An average topographic flattening of 11.4 diopters and pachymetric thinning of 50 microns at the center of the treatment zone were observed by 6 weeks following treatment. The average diameter of the zone of flattening was 5.1 mm at 6 weeks and 6.0 mm at 12 weeks postoperatively. At 6 months postoperatively, the average central corneal power was unchanged, while the average central thickness had returned to the preoperative value. A mild subepithelial haze was detected at 1 month following IPRK, which was barely perceptible at the second through sixth months. Regression of the topographic effects of PRK was not observed. CONCLUSION: Intrastromal PRK with the Nd-YLF picosecond laser produces central flattening of the cat cornea with barely perceptible stromal haze and no regression of topographic effect after 6 months in the absence of treatment with anti-inflammatory agents.

Mathematical models of picosecond laser keratomileusis for high myopia.

PURPOSE: Picosecond laser keratomileusis (PLK) is a refractive surgical procedure in which an intrastromal lenticle is created using the Nd:YLF picosecond laser and removed by lifting the anterior flap in the cornea. The purpose of this study was to assess the predictability of picosecond laser keratomileusis for high myopia. METHODS: The results from a recent clinical series of non-sighted patients who had undergone picosecond laser keratomileusis with the Nd:YLF picosecond laser were retrospectively simulated with two mathematical models. In one model, the change in corneal curvature resulting from the collapse of a lenticular intrastromal cavity was predicted from a geometric analysis. A second model was developed using the finite element method. The results of both predictive models were compared to the clinical results at 1 and 6 months following surgery. RESULTS: An average -13.30 D of flattening (range, -6.80 to -20.50 D) was measured at 6 months. The geometric model predicted an average correction of -23.10 D (range, -18.00 to -34.00 D), whereas the finite element model predicted an average curvature change of -17.40 D (range, -14.50 to -22.50 D). An average decrease in central pachymetry of 91 microm was measured at 6 months, as compared to a predicted decrease in thickness of 112 microm. CONCLUSIONS: The outcome of picosecond laser keratomileusis for high myopia predicted using a finite element based model shows greater change in the corneal curvature and corneal thickness compared to clinical results. The relatively thick lenticules that are removed for high myopia cause considerable deformational changes in the cornea, which preclude the use of a purely geometric/optical approach for predicting the resultant corneal curvature changes.

The picosecond laser for nonmechanical laser in situ keratomileusis.

PURPOSE: To introduce the clinical use of the Nd:YLF picosecond laser as a nonmechanical microkeratome. METHODS: A commercially available Nd:YLF picosecond laser (25 microJ/pulse, 30 psec, 1053 nm) was used to deliver intrastromal pulses of focused high power laser light to generate a flap for laser in situ keratomileusis (LASIK) and perform picosecond laser keratomileusis (PLK) in two partially sighted patients with high myopia. RESULTS: Case #1: A 6 mm, 150 microns flap for LASIK was successfully created and the underlying stroma treated with an excimer laser for a target correction of -15.00 D of myopia. Good corneal clarity and a refractive change of -14.00 D was recorded 2 months postoperatively. Case #2: Both eyes of a high myope (-22.00 D right eye, -21.50 D left eye) underwent picosecond laser keratomileusis, removing a 3.2 mm diameter lenticule of 120 microns thickness under a 200 microns flap. Postoperative refraction was -2.00 D at 7 months in the right eye and -0.75 D at 2 months in the left eye with improvement of spectacle-corrected visual acuity from 20/200 to 20/70 in each eye. CONCLUSION: The Nd:YLF picosecond laser can be safely used in creating a corneal flap for LASIK and in performing picosecond laser keratomileusis for high myopia. Future refinements in the laser will include a larger flap diameter and femtosecond pulsing capability.

Ultrastructure of picosecond laser intrastromal photodisruption.

PURPOSE: To investigate the ultrastructure of the corneal stroma after picosecond intrastromal photodisruption with a neodymium-doped yttrium-lithium-fluoride (Nd:YLF) laser. METHODS: We performed picosecond intrastromal photodisruption on six human eye-bank eyes using a lamellar technique. Thirty picosecond pulses at 1000 Hz and 20 to 25 mJ per pulse were placed in an expanding spiral pattern, the pulses separated by 15 microns. Three layers were placed in the anterior stroma, separated from each other by 15 microns. In addition, intrastromal radial and arcuate incisions were generated in two living rabbit eyes in a plane perpendicular to the corneal surface. After the procedure, the corneas were processed for scanning and transmission electron microscopy. RESULTS: Scanning electron microscopy of the eye-bank eyes demonstrated multiple, coalescing intrastromal cavities forming a layer oriented parallel to the corneal surface. These cavities had smooth inner walls. Transmission electron microscopy demonstrated tissue loss surrounding some cavities, with the terminated ends of collagen fibrils clearly evident. Other cavities were formed by separation of lamellae, with little evidence of tissue loss. A pseudomembrane was present along the margin of some cavities. Although there was occasional underlying tissue disruption along the border of a cavity, there was no evidence of thermal damage or tissue necrosis. The perpendicular photodisruptions demonstrated intrastromal cleavage of corneal collagen similar to diamond-knife incisions, with the exception of intact overlying Bowman's and epithelial layers. CONCLUSION: Intrastromal photodisruption with a Nd:YLF picosecond laser induced no thermal necrosis or coagulative change in the region of tissue interaction. Lamellar intrastromal photodisruption demonstrated both tissue loss and lamellar separation when performed with the current treatment parameters, possibly limiting ablation efficiency and predictability.

Lamellar refractive surgery with scanned intrastromal picosecond and femtosecond laser pulses in animal eyes.

PURPOSE: To evaluate the use of scanned intrastromal picosecond and femtosecond laser pulses in lamellar refractive surgical procedures. METHODS: Intrastromal corneal photodisruption was performed in fresh porcine and primate cadaver eyes with a solid-state femtosecond laser. Laser pulses were focused 150 to 200 microns below the epithelial surface and scanned in a spiral pattern to create a plane. A flap was made by scanning an arc pattern from the plane of the spiral to the surface of the cornea. Tissue plane separation was graded using a standard scale, while internal surfaces were analyzed by scanning electron microscopy. Comparison was made to a picosecond laser system using the same delivery system device. Creation of a stromal lenticule for in situ keratomileusis was also demonstrated and compared with both laser systems. RESULTS: For femtosecond pulses, tissue separation was achieved best with pulse energies from 4 to 8 microJ and spot separations from 10-15 microns. Picosecond pulses accomplished less complete separations with pulse energies of 25 microJ and spot separations from 10 to 20 microns. Surface quality corresponded to dissection results, with high-grade dissections resulting in a smooth surface appearance, versus a more irregular surface for low-grade dissections. Although high-grade dissections could be created with picosecond pulses (with optimal parameters) in ex vivo porcine eyes, only femtosecond parameters produced similar results in ex vivo primate eyes. CONCLUSION: In contrast to previous attempts using picosecond lasers which require additional mechanical dissection, high precision lamellar refractive surgery may be practical with femtosecond laser pulses.

Clinical analysis of the neodymium:YLF picosecond laser as a microkeratome for laser in situ keratomileusis. Partially Sighted Eye Study.

PURPOSE: To evaluate the use of a picosecond neodymium:YLF (Nd:YLF) laser as a nonmechanical intrastromal microkeratome. SETTING: Universita Cattolica del Sacro Cuore, Rome, Italy. METHODS: An intrastromal spiral disc pattern of picosecond laser pulses was used to create a corneal flap for laser in situ keratomileusis (LASIK) in 14 partially sighted eyes. RESULTS: Flaps with a 6.0 mm diameter and 180 to 200 microns depth were successfully created in most cases. The underlying stroma was treated with a Lambda Physik excimer laser using a 3.5 to 4.5 mm optical zone. Patients were divided into 3 groups for target corrections of 5.0, 10.0, and 15.0 diopters of myopia. Good corneal clarity and refractive undercorrection were recorded in each group 6 months postoperatively. The undercorrection was due in part to the limited optical zone of the laser's delivery system. Some flap decentration was noted. CONCLUSION: This pilot study indicates that the Nd:YLF picosecond laser may be clinically applied for creating corneal flaps for LASIK. Further refinements of the laser delivery system will include enlargement of the flap diameter and improvements in flap centration. The use of a femtosecond laser may expand the capabilities and precision of this technology.

Mixed species flocking of tits (Parus spp.): a field experiment.

We tested two general models of flocking behaviour, namely the antipredation model and foraging efficiency model on mixed-species tit flocks (Parus spp.). After food addition the size of mixed-species flocks was significantly less than in the control samples. In the presence of extra food significantly more birds were observed either in monospecific flocks or solitary, than during the control observations. In the presence of a living predator the birds foraged in larger mixed-specifies flocks than during the control observations. In addition, the social behaviour of Great Spotted Woodpecker, Middle Spotted Woodpecker and Nuthatch shifted to mixed-specific flocking. The size of monospecific flocks was independent of both treatments. The density of birds increased significantly after food addition, while in the predator presence the birds tended to leave the forest. These results support the view that both the antipredation model and foraging efficiency model seem to be valid for mixed-species flocking. However, in the case of monospecific flocks, the territory maintenance could be the most important factor.

Ultra-short pulse (femtosecond) laser surgery: initial use in LASIK flap creation.

The highly localized tissue effects of low energy femtosecond duration (ultrashort) laser pulses may be used to create three-dimensional intrastromal resections with micron precision and minimized collateral tissue damage. A surgical laser system that produces and delivers such pulses has been developed and tested clinically for creation of a corneal flap in LASIK. Expanded evaluation of this technology in this and additional keratorefractive applications is currently underway.

An in vivo model of femtosecond laser intrastromal refractive surgery.

BACKGROUND AND OBJECTIVE: To develop an animal model for evaluation of femtosecond laser intrastromal refractive surgery. METHODS: Intrastromal photodisruption was performed in New Zealand Albino rabbits using a femtosecond laser system. This surgical pattern consisted of a 100 microm-tick pyramid of laser pulses starting 180 microm below the corneal surface. Animals underwent serial slit lamp examinations and corneal thickness measurements at 1,3,7,14, and 28 days, then monthly up to 1 year. RESULTS: Approximately 70 microm of central corneal thinning were seen at 1 week, remaining stable up to 7 months. CONCLUSIONS: Intrastromal photodisruption with femtosecond lasers produced consistent changes in corneal thickness without loss of corneal transparency. These changes were more stable than those produced with excimer laser procedures in a similar animal model.

Distribution of extracellular matrix macromolecules in the vestibular nuclei and cerebellum of the frog, Rana esculenta.

The axons of transected and re-apposed vestibulocochlear nerve of the frog, in contrast to mammalian species, regenerate and establish functional contacts within their original termination areas of the vestibular nuclear complex and the cerebellum. The lack of regenerative capability of the mammalian central nervous system (CNS) is partially attributed to various extracellular matrix (ECM) molecules, such as chondroitin sulfate proteoglycans (CSPG) and tenascin-R (TN-R), which exert inhibition on axon regeneration. In contrast to these molecules, hyaluronan (HA) was reported to be permissive for CNS regeneration. Using histochemical and immunohistochemical methods, we investigated the distribution pattern of these molecules in the medial (MVN), lateral (LVN), superior and descending vestibular nuclei and the cerebellum of the frog and detected regional differences in the organization of the ECM. In the vestibular nuclear complex, pericellular condensation of the ECM, the perineuronal nets (PNNs) were recognizable in the LVN and MVN and were positive only for HA. The neuropil of the vestibular nuclei showed either a diffuse appearance with varying intensity of reactions, or dots and ring-like structures, which may represent the perinodal ECM of the vestibular fibers. In the cerebellum, indistinct PNNs that were only labeled for HA were present in the granular layer. Our findings suggest that the HA-rich, but CSPG and TN-R-free PNNs may be associated with the high degree of plasticity and regenerative potential of the amphibian vestibular system.

Structural and morphometric comparison of the molar teeth in pre-eruptive developmental stage of PACAP-deficient and wild-type mice.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a pleiotropic neuropeptide with widespread distribution. It plays pivotal role in neuronal development. PACAP-immunoreactive fibers have been found in the tooth pulp, and recently, it has been shown that PACAP may also play a role in the regeneration of the periodontium after luxation injuries. However, there is no data about the effect of endogenous PACAP on tooth development. Ectodermal organogenesis including tooth development is regulated by different members of bone morphogenetic protein (BMP), fibroblast growth factor (FGF), hedgehog (HH), and Wnt families. There is also a growing evidence to support the hypothesis that PACAP interacts with sonic hedgehog (SHH) receptor (PTCH1) and its downstream target (Gli1) suggesting its role in tooth development. Therefore, our aim was to study molar tooth development in mice lacking endogenous PACAP. In this study morphometric, immunohistochemical and structural comparison of molar teeth in pre-eruptive developmental stage was performed on histological sections of 7-day-old wild-type and PACAP-deficient mice. Further structural analysis was carried out with Raman microscope. The morphometric comparison of the 7-day-old samples revealed that the dentin was significantly thinner in the molars of PACAP-deficient mice compared to wild-type animals. Raman spectra of the enamel in wild-type mice demonstrated higher diversity in secondary structure of enamel proteins. In the dentin of PACAP-deficient mice higher intracrystalline disordering in the hydroxyapatite molecular structure was found. We also obtained altered SHH, PTCH1 and Gli1 expression level in secretory ameloblasts of PACAP-deficient mice compared to wild-type littermates suggesting that PACAP might play an important role in molar tooth development and matrix mineralization involving influence on SHH signaling cascade.

Cellular and ultra structural evidence for cytoskeletal localization of prolyl endopeptidase-like protein in neurons.

The biochemical properties and subcellular localization of prolyl endopeptidase (PREP) in brain are well characterized and its implications in the realization of cognitive processes and in the pathogenesis of neurodegenerative disorders are a matter of intensive investigation. In contrast, very little is known about its homolog, the PREP-like protein (PREPL). In order to obtain initial hints about the involvement of PREPL in physiological processes, a differential proteomic screen was performed with human skin fibroblasts from controls and patients with PREPL deficiency (hypotonia-cystinuria syndrome). The majority of affected proteins represented cytoskeletal proteins, including caldesmon, tropomyosin α3 chain, lamin A, ß-actin, γ-actin, vimentin and zyxin. Therefore, the analysis of PREPL subcellular localization by confocal laser scanning and electron microscopy in mouse neurons was focused on the cytoskeleton. The co-localization of PREPL with cytoskeletal marker proteins such as ß-actin and microtubulin-associated protein-2 was observed, in addition to the presence of PREPL within Golgi apparatus and growth cones. In the mouse brain, PREPL is neuronally expressed and highly abundant in neocortex, substantia nigra and locus coeruleus. This mirrors to some extent the distribution pattern of PREP and points toward redundant functions of both proteins. In the human neocortex, PREPL immunostaining was found in the cytoplasm and in neuropil, in particular of layer V pyramidal neurons. This staining was reduced in the neocortex of Alzheimer's disease (AD) patients. Moreover, in AD brains, PREPL immunoreactivity was observed in the nucleus and in varicose neuritic processes. Our data indicate physiological functions of PREPL associated with the cytoskeleton, which may be affected under conditions of cytoskeletal degeneration.

Comparative assay of fluorescent antibody test results among twelve European National Reference Laboratories using various anti-rabies conjugates.

Twelve National Reference Laboratories (NRLs) for rabies have undertaken a comparative assay to assess the comparison of fluorescent antibody test (FAT) results using five coded commercial anti-rabies conjugates (Biorad, Bioveta, Fujirebio, Millipore, and SIFIN conjugates). Homogenized positive brain tissues infected with various lyssavirus species as well as negative samples were analyzed blindly using a standardized FAT procedure. Conjugates B, C, D, and E were found to be significantly more effective than conjugate A for GS7 (French RABV) diluted samples (1/8 and 1/100) while the frequency of concordant results of conjugates C and D differ significantly from conjugates A, B and E for CVS 27. For detection of EBLV-1 strains, conjugates C and D also presented a significantly lower frequency of discordant results compared to conjugates A, B and E. Conjugates B, C and D were found to be significantly more effective than conjugates E and A for EBLV-2 and ABLV samples. In view of these results, conjugates C and D set themselves apart from the others and appeared as the most effective of this 5-panel conjugates. This study clearly demonstrates that the variability of conjugates used by National Reference Laboratories can potentially lead to discordant results and influence assay sensitivity. In case of false negative results this could have a dramatic impact if the animal under investigation is responsible for human exposure. To avoid such situations, confirmatory tests should be implemented.