Blood Analysis of Laboratory Macaca mulatta Used for Neuroscience Research: Investigation of Long-Term and Cumulative Effects of Implants, Fluid Control, and Laboratory Procedures.

The nonhuman primate (NHP) constitutes an extraordinarily important model in neuroscience research for understanding the neuronal underpinnings of perceptual, motor, cognitive, and executive functions of the primate brain, and to study the physiological causes, effects, and potential treatments of brain disorders. Because of their cognitive capabilities, NHPs receive special attention in animal welfare regulations around the world, and their well-being is a benchmark for the evaluation, monitoring, and refinement of experimental procedures. As a consequence, many typical neuroscientific procedures are considered only mildly severe by animal welfare boards. There is, however, an ongoing debate about possible long-term and cumulative effects. Because of a lack of longitudinal data, it is unclear whether mildly severe procedures may cause more significant harm on the long-term, and to what extent they may impact animal well-being and healthiness over time. We here make use of a database of blood samples drawn over a period of 15 years from 39 rhesus monkeys (Macaca mulatta) to address the issue of long-term, cumulative effects of neuroscientific procedures. A careful analysis of indicative primate blood markers for chronic inflammation, hydration status, and stress levels, their comparison to baseline values from both the same animals and the literature, and evaluation of additional hematologic, physiological, and behavioral parameters did not provide support for the notion of long-term, cumulative effects on the monkeys' healthiness and well-being. The results may serve the community as a reference for the severity assessment of neuroscientific experiments involving NHPs.

Comparative Study of Optical Markers to Assess Bait System Efficiency Concerning Vaccine Release in the Oral Cavity of Dogs.

Oral vaccination of dogs against rabies has the potential to achieve mass coverage and thus deplete the virus of its most important reservoir host species. There is, however, no established non-invasive method to evaluate vaccine release in the oral cavity, following bait ingestion. In this study, two pre-selected marker methods in conjunction with their acceptance were assessed in local Thai dogs. Shelter dogs (n = 47) were offered one of four randomized bait formulations; bait type A-, containing Green S (E142) in a fructose solution; type B-, containing Patent Blue V (E131) in a fructose solution; type C-, containing the medium used for delivery of oral rabies vaccine in baits commercially produced; and type D-, containing denatonium benzoate, which was to serve as the negative control, due to its perceived bitterness. Patent Blue V was found to possess overall stronger dyeing capacities compared to Green S. Furthermore, there was no significant difference in the acceptance or bait handling of Patent Blue V baits compared to those containing the oral rabies vaccine medium alone, suggesting the potential use of this dye as a surrogate for rabies vaccine when testing newly developed bait formats.

Helminth infections of wild European gray wolves (Canis lupus Linnaeus, 1758) in Lower Saxony, Germany, and comparison to captive wolves.

This study aimed to investigate the endoparasite fauna of wild European gray wolves, which are currently recolonizing Germany. In total, 69 fecal samples of wild wolves were collected in Lower Saxony, Germany, from 2013 to 2015, analyzed by the sedimentation-flotation and McMaster techniques and compared to previous results on captive European Gray wolves living in zoological gardens in Germany. In addition to coproscopy, taeniid-positive samples from wild as well as captive wolves were differentiated by amplification and sequencing of small subunit ribosomal RNA (SSU rRNA) and NADH dehydrogenase 1 (nad1) gene fragments. Missing Taenia krabbei SSU rRNA reference sequences were generated from two T. krabbei specimens. Overall, 60.87% (42/69) of wild wolve samples were microscopically positive for at least one of seven egg types. Capillaria/Eucoleus spp. showed the highest frequency (31.88% [22/69]), followed by Taeniidae (21.74% [15/69]), Ancylostomatidae (20.29% [14/69]), Alaria alata (15.94% [11/69]), Toxocara canis (13.04% [9/69]), and Toxascaris leonina and Trichuris vulpis (each 5.80% [4/69]). Amplification of SSU rRNA was successful for 7/15 Taeniidae-positive samples from wild and 20/39 samples from captive wolves, revealing T. hydatigena in two and 14 samples, respectively. Taenia krabbei was detected in two further samples of wild and three samples of captive wolves, while for the remaining samples, no differentiation between T. serialis/T. krabbei was possible. Echinococcus spp. were not detected. Sequence comparisons revealed that the SSU rRNA gene fragment was not suitable to differentiate between T. serialis and T. krabbei. Therefore, the use of this fragment alone cannot be recommended for species identification in future studies.

Modular AUV System with Integrated Real-Time Water Quality Analysis.

This paper describes the concept, the technical implementation and the practical application of a miniaturized sensor system integrated into an autonomous underwater vehicle (AUV) for real-time acquisition of water quality parameters. The main application field of the presented system is the analysis of the discharge of nitrates into Norwegian fjords near aqua farms. The presented system was developed within the research project SALMON (Sea Water Quality Monitoring and Management) over a three-year period. The development of the sensor system for water quality parameters represented a significant challenge for the research group, as it was to be integrated in the payload unit of the autonomous underwater vehicle in compliance with the underwater environmental conditions. The German company -4H- JENA engineering GmbH (4HJE), with experience in optical in situ-detection of nutrients, designed and built the measurement system. As a carrier platform, the remotely operated vehicle (ROV) "CWolf" from Fraunhofer-Institut für Optronik, Systemtechnik und Bildauswertung - Institutsteil Angewandte Systemtechnik (IOSB-AST) modified to an AUV was deployed. The concept presented illustrates how the measurement system can be integrated easily into the vehicle with a minimum of hard- and software technical interfaces.

Helminth Fauna in Captive European Gray Wolves (Canis lupus lupus) in Germany.

Captive as well as free-ranging wolves, which are currently recolonizing Germany, may harbor a variety of gastrointestinal parasites. This study investigated endoparasites in captive European gray wolves (Canis lupus lupus) using coproscopical methods. Fecal samples were collected monthly between October 2012 and November 2013 from 18 wolf enclosures in 14 German zoological gardens, representing 72 individual wolves. In total, 1,041 fecal samples including 26 bulk samples were analyzed by the sedimentation and flotation method. The most frequently detected egg morphotypes included five nematodes [Ancylostomatidae (Ancylostoma or Uncinaria spp.), Toxocara canis, Toxascaris leonina, Trichuris vulpis, and Capillaria/Eucoleus spp.], one cestode (Taeniidae) and one trematode (Alaria alata). 44.76% of all samples were positive for at least one of these egg morphotypes. Overall, Ancylostomatidae showed the highest frequency (30.84% of all samples), followed by Capillaria/Eucoleus spp. (19.88%), Toxocara canis (5.19%), taeniids (3.75%), Trichuris vulpis and Alaria alata (3.65% each), and Toxascaris leonina (1.25%). As fecal samples were collected from the environment and could not be assigned to individual wolves, sample results were combined per zoo and month. General linear mixed models were employed to analyze the effect of season and management factors on the occurrence of Ancylostomatidae, Capillaria/Eucoleus spp., Toxocara canis and taeniids. No statistically significant effect of season was found, whereas anthelmintic treatment negatively affected Ancylostomatidae egg excretion. Detected parasites and their prevalences are comparable to previous studies on wolf parasitism conducted elsewhere in Europe. As many of the most prevalent helminths are of zoonotic importance, routine anthelmintic treatment of captive wolves should be recommended.

Immunophenotyping of immune cell populations in the raccoon (Procyon lotor).

The raccoon (Procyon lotor) is a highly adaptable carnivore that has rapidly conquered Europe over the last decades and represents a potential candidate as pathogen reservoir, bearing the risk for transmission of infectious agents, as zoonosis or spill-over, to other wild life and domestic animals and man. Comprehensive investigations of infectious diseases in raccoons require a detailed knowledge of the participating immune cell populations. To close this gap of knowledge, various antibodies were tested for cross-reactivity with leukocytes in lymphoid organs and peripheral blood of raccoons using immunohistochemistry and flow cytometry, respectively. Eight out of 16 antibodies, directed against CD3, CD79α, Pax-5, IgG, CD44, MHC class II, myeloid/histiocyte antigen (MAC387), and Iba-1 exhibited a specific immunoreaction with cells in distinct anatomical compartments in formalin-fixed paraffin-embedded lymphoid tissues. Flow cytometric analysis revealed that 7 out of 18 antibodies directed against CD11c, CD14, CD21, CD44, CD79α, MHC class I and II cross-reacted with peripheral blood-derived raccoon leukocytes. Summarized, the usefulness of several cross-reacting antibodies was determined for the characterization of raccoon immune cells in immunohistochemistry and flow cytometry, offering the opportunity to study the raccoon immune system under normal and diseased conditions.

Fatal epizootic equine herpesvirus 1 infections in new and unnatural hosts.

In a zoological collection, four black bears (Ursus americanus) died from neurological disease within six months. Independently in a geographically different zoo, two Thomson's gazelles (Eudorcas thomsoni) and 18 guinea pigs (Cavia porcellus f. dom.) suffered from neurological disorders. In addition, guinea pigs showed abortions and stillbirths. All affected animals displayed a non suppurative meningoencephalitis with intranuclear inclusion bodies. Immunohistology demonstrated equine herpes virus antigen and ultrastructurally herpes viral particles were detected. Virus isolation and molecular analysis identified neurotropic equine herpesvirus (EHV) 1 strains in both epizootics. There is serological evidence of a possible virus transmission from other equids to the affected animals. Cross-species transmission of EHV-1 should be considered in the management of captive wild equids and ungulates, particularly with respect to fatal disease in irreplaceable species.

Epizootic fatal amebiasis in an outdoor group of Old World monkeys.

BACKGROUND: Entamoeba (E.) histolytica is an obligate parasite of humans and non-human primates. METHODS: This report describes the pathomorphological, immunohistological, and microbiological findings of fatal E. histolytica infection in two mantled guerezas (Colobus guereza) and one Hanuman langur (Semnopithecus entellus) from an epizootic outbreak of amebiasis in an open-range recreation park. RESULTS: Pathomorphological examination revealed multifocal necrotizing and granulomatous hepatitis with intralesional protozoan trophozoites in all three cases. In addition, necrotizing and ulcerative gastritis was detected in both mantled guerezas. Furthermore, oligofocal acute pulmonary embolization was detected in one of these cases. No extra-hepatic lesions were observed in the Hanuman langur. Immunohistological examination confirmed the etiologic diagnosis of E. histolytica-induced lesions. CONCLUSIONS: Although E. histolytica is a rarely diagnosed pathogen in Western European countries, veterinarians and animal keepers involved in handling and care taking of non-human primates should be aware of the potential threat caused by this zoonotic parasite.

Quality criteria were proposed for measurement properties of health status questionnaires.

OBJECTIVES: Recently, an increasing number of systematic reviews have been published in which the measurement properties of health status questionnaires are compared. For a meaningful comparison, quality criteria for measurement properties are needed. Our aim was to develop quality criteria for design, methods, and outcomes of studies on the development and evaluation of health status questionnaires. STUDY DESIGN AND SETTING: Quality criteria for content validity, internal consistency, criterion validity, construct validity, reproducibility, longitudinal validity, responsiveness, floor and ceiling effects, and interpretability were derived from existing guidelines and consensus within our research group. RESULTS: For each measurement property a criterion was defined for a positive, negative, or indeterminate rating, depending on the design, methods, and outcomes of the validation study. CONCLUSION: Our criteria make a substantial contribution toward defining explicit quality criteria for measurement properties of health status questionnaires. Our criteria can be used in systematic reviews of health status questionnaires, to detect shortcomings and gaps in knowledge of measurement properties, and to design validation studies. The future challenge will be to refine and complete the criteria and to reach broad consensus, especially on quality criteria for good measurement properties.

Direct observation of intraparticle equilibration and the rate-limiting step in adsorption of proteins in chromatographic adsorbents with confocal laser scanning microscopy.

The adsorption of different proteins in a single biospecific and hydrophobic adsorbent particle for preparative protein chromatography has been observed directly by confocal laser scanning microscopy as a function of time at a constant bulk concentration c(b). The bulk concentration was in the non-linear part of the adsorption isotherm. At all times the concentration of free protein at the particle surface was almost equal to the bulk content indicating that external mass transfer resistance is not rate limiting for the adsorption under these conditions. Inside the particles a distinct maximum in adsorbed and free protein concentration that moved inside to a distance of approximately 0.2 R (R particle radius) from the particle surface, was observed. This is due to a decreasing solid-phase density and adsorptive capacity in the particle between 0.8 R and R indicating that the fraction of macropores (or void space) is larger in the outer than in the inner part of the adsorbent particles. By increasing the bulk concentration by a factor of 10 the equilibration time was reduced by about the same magnitude. This is in agreement with the concentration dependence of the effective pore diffusion coefficient D(p,eff)=D(p)/[epsilon(p)[1+nK/(K +c)(2)]] derived from the mass conservation relations describing the adsorption process. The time dependence protein adsorption up to approximately 90% of the equilibration value q* could be described by a bilinear free driving force model. The rapid equilibration in the outer part of the particle with a half-life time of approximately 100 s in the studied systems accounted for 0.3-0.4 q*. The slower equilibration with a up to ten times longer half-life time, was the adsorption in the inner part of the particle that outside 0.5 R accounts for 0.5-0.6 q*. These data were compared with literature data for batch adsorption of proteins in biospecific, hydrophobic and ion-exchange adsorbents. They could also be described by a bilinear free driving force model, with about the same quantitative results as obtained for similar conditions in the single particle experiments. The static adsorption parameters, maximum binding site concentration n, and dissociation constant for the protein binding to a binding site K, were determined from Scatchard plots. For the same protein-adsorbent system the plots changed from linear to non-linear with increasing n. This change occurred when the average distance between adjacent binding sites become of the same order of magnitude as the size of the binding site or adsorbed protein. This causes a shielding of free binding sites increasing with n and the concentration of adsorbed protein, yielding a concentration dependence in K. These results show that for a high throughput and rapid adsorption in preparative chromatography, the adsorption step should be carried out in the non-linear part of the adsorption isotherm with concentrations up to c(b) where q*/c(b)>/=10 to obtain high protein recoveries. To avoid tailing due to the flow of adsorbed proteins in the inner part of the particles further into the particles at the start of the desorption, and to speed up desorption rates, protein adsorption in the particle within 0.5 R from the particle center should be avoided. This requires the further development of suitable pellicular particles for preparative protein chromatography that meet this requirement.