Serving science and conservation: The biological materials request protocol of the New York Zoological Society

Biological materials obtained during routine veterinary treatment and necropsies on zoo and aquarium animals provide opportunities to study a wide variety of phenomena, many of which have important implications for wildlife conservation and captive animal management. Collection and shipment of biological materials require extensive coordination, and few zoological institutions have developed detailed, standardized protocols to facilitate these efforts. The value of using biological materials is reviewed and a set of policies and guidelines are outlined for their collection, preservation, and shipment. Several related issues are discussed, including health hazards and liability issues, permit requirements, and animal welfare considerations.     

Chicken- and duck-associated Bacteroides–Prevotella genetic markers for detecting fecal contamination in environmental water

Bacteroides–Prevotella group is one of the most promising targets for detecting fecal contamination in water environments, principally due to its host-specific distributions and high concentrations in feces of warm-blooded animals. We developed real-time PCR assays for quantifying chicken/duck-, chicken-, and duck-associated Bacteroides–Prevotella 16S rRNA genetic markers (Chicken/Duck-Bac, Chicken-Bac, and Duck-Bac). A reference collection of DNA extracts from 143 individual fecal samples and wastewater treatment plant influent was tested by the newly established markers. The quantification limits of Chicken/Duck-Bac, Chicken-Bac, and Duck-Bac markers in environmental water were 54, 57, and 12 copies/reaction, respectively. It was possible to detect possible fecal contaminations from wild ducks in environmental water with the constructed genetic marker assays, even though the density of total coliforms in the identical water samples was below the detection limit. Chicken/Duck-Bac marker was amplified from feces of wild duck and chicken with the positive ratio of 96 and 61 %, respectively, and no cross-reaction was observed for the other animal feces. Chicken-Bac marker was detected from 70 % of chicken feces, while detected from 39 % of cow feces, 8.3 % of pig feces, and 12 % of swan feces. Duck-Bac marker was detected from 85 % of wild duck feces and cross-reacted with 31 % of cow feces. These levels of detection specificity are common in avian-associated genetic markers previously proposed, which implies that there is a practical limitation in the independent application of avian-associated Bacteroides–Prevotella 16S rRNA genetic markers and a combination with other fecal contamination markers is preferable for detecting fecal contamination in water environments.     

Impact of zoo visitors on the fecal cortisol levels and behavior of an endangered species: Indian blackbuck (Antelope cervicapra L.)

This study investigated behavioral activities (resting, moving, aggressive, social, and reproductive behavior) and fecal cortisol levels in 8 individually identified adult male blackbucks during periods of varying levels of zoo visitors (zero, low, high, and extremely high zoo visitor density). This study also elucidated whether zoo visitor density could disturb nonhuman animal welfare. This study analyzed fecal cortisol from the samples of blackbuck by radioimmunoassay and found significant differences (p < .05) for time the animals devoted to moving, resting, aggressive, reproductive, and social behavior on days with high and extremely high levels of zoo visitors. The ANOVA with Duncan's Multiple Range Test test showed that the fecal cortisol concentration was higher (p < .05) during the extremely high (137.30 ± 5.88 ng/g dry feces) and high (113.51 ± 3.70 ng/g dry feces) levels of zoo visitor density. The results of the study suggest that zoo visitor density affected behavior and adrenocortical secretion in Indian Blackbuck, and this may indicate an animal welfare problem.     

Impact of Zoo Visitors on the Fecal Cortisol Levels and Behavior of an Endangered Species: Indian Blackbuck (Antelope cervicapra L.)

This study investigated behavioral activities (resting, moving, aggressive, social, and reproductive behavior) and fecal cortisol levels in 8 individually identified adult male blackbucks during periods of varying levels of zoo visitors (zero, low, high, and extremely high zoo visitor density). This study also elucidated whether zoo visitor density could disturb nonhuman animal welfare. This study analyzed fecal cortisol from the samples of blackbuck by radioimmunoassay and found significant differences (p < .05) for time the animals devoted to moving, resting, aggressive, reproductive, and social behavior on days with high and extremely high levels of zoo visitors. The ANOVA with Duncan's Multiple Range Test test showed that the fecal cortisol concentration was higher (p < .05) during the extremely high (137.30 ± 5.88 ng/g dry feces) and high (113.51 ± 3.70 ng/g dry feces) levels of zoo visitor density. The results of the study suggest that zoo visitor density affected behavior and adrenocortical secretion in Indian Blackbuck, and this may indicate an animal welfare problem.     

What free‐ranging animals do at the zoo: a study of the behavior and habitat use of opossums (Didelphis virginiana) on the grounds of the St. Louis Zoo

Studies of urban wildlife are important because they can give us insight into both how animals adapt to novel environments and how some species survive and prosper in human‐dominated landscapes. Urban zoological institutions provide an ideal setting for research on such species. We report on a study of the behavior and ecology of opossums (Didelphis virginiana) on the grounds of the St. Louis Zoological Park. We used radio tracking to investigate the movement patterns and den site use of opossums on the grounds of the zoo and compared the results to data available for opossums elsewhere. We find that urban opossums in the St. Louis zoo have smaller home ranges and move shorter distances than their rural counterparts. We suggest that increased food availability and decreased risk of predation might explain such differences, and we suspect that conditions are even more favorable to opossums in a zoological setting than in cities in general. Our findings illustrate that there is much to be learned from scientific study of the free‐living animals found on zoological park grounds. Zoo Biol 00:1–17, 2005. © 2005 Wiley‐Liss, Inc.     

Behavioral monitoring in zoos and aquariums: a tool for guiding husbandry and directing research

Behavioral monitoring is the scientific collection of animal behavior data to understand normal patterns of behavior and changes in these patterns. This tool is underutilized in the zoo industry although it can be an effective indicator of many potential problems that compromise zoo animal well-being. We suggest that a behavioral monitoring program should be a core component of a zoological institution's care program. We detail the benefits of such a program and describe its components. We provide guidelines for implementing such a program and make recommendations that will help institutions to employ behavioral monitoring programs with reasonable expense. We argue that the benefits of such a program, primarily increased detection of rising or potential problems, far outweigh the minor costs of implementation. Zoo Biol 28:35–48, 2009. © 2008 Wiley-Liss, Inc.     

Evaluation of two library-independent microbial source tracking methods to identify sources of fecal contamination in French estuaries

In order to identify the origin of the fecal contamination observed in French estuaries, two library-independent microbial source tracking (MST) methods were selected: (i) Bacteroidales host-specific 16S rRNA gene markers and (ii) F-specific RNA bacteriophage genotyping. The specificity of the Bacteroidales markers was evaluated on human and animal (bovine, pig, sheep, and bird) feces. Two human-specific markers (HF183 and HF134), one ruminant-specific marker (CF193'), and one pig-specific marker (PF163) showed a high level of specificity (>90%). However, the data suggest that the proposed ruminant-specific CF128 marker would be better described as an animal marker, as it was observed in all bovine and sheep feces and 96% of pig feces. F RNA bacteriophages were detected in only 21% of individual fecal samples tested, in 60% of pig slurries, but in all sewage samples. Most detected F RNA bacteriophages were from genotypes II and III in sewage samples and from genotypes I and IV in bovine, pig, and bird feces and from pig slurries. Both MST methods were applied to 28 water samples collected from three watersheds at different times. Classification of water samples as subject to human, animal, or mixed fecal contamination was more frequent when using Bacteroidales markers (82.1% of water samples) than by bacteriophage genotyping (50%). The ability to classify a water sample increased with increasing Escherichia coli or enterococcus concentration. For the samples that could be classified by bacteriophage genotyping, 78% agreed with the classification obtained from Bacteroidales markers.     

Concentrations of pathogens and indicators in animal feces in the Sydney watershed

A fecal analysis survey was undertaken to quantify animal inputs of pathogenic and indicator microorganisms in the temperate watersheds of Sydney, Australia. The feces from a range of domestic animals and wildlife were analyzed for the indicator bacteria fecal coliforms and Clostridium perfringens spores, the pathogenic protozoa Cryptosporidium and Giardia, and the enteric viruses adenovirus, enterovirus, and reovirus. Pathogen and fecal indicator concentrations were generally higher in domestic animal feces than in wildlife feces. Future studies to quantify potential pathogen risks in drinking-water watersheds should thus focus on quantifying pathogen loads from domestic animals and livestock rather than wildlife.     

Development of a swine-specific fecal pollution marker based on host differences in methanogen mcrA genes

The goal of this study was to evaluate methanogen diversity in animal hosts to develop a swine-specific archaeal molecular marker for fecal source tracking in surface waters. Phylogenetic analysis of swine mcrA sequences compared to mcrA sequences from the feces of five animals (cow, deer, sheep, horse, and chicken) and sewage showed four distinct swine clusters, with three swine-specific clades. From this analysis, six sequences were chosen for molecular marker development and initial testing. Only one mcrA sequence (P23-2) showed specificity for swine and therefore was used for environmental testing. PCR primers for the P23-2 clone mcrA sequence were developed and evaluated for swine specificity. The P23-2 primers amplified products in P23-2 plasmid DNA (100%), pig feces (84%), and swine waste lagoon surface water samples (100%) but did not amplify a product in 47 bacterial and archaeal stock cultures and 477 environmental bacterial isolates and sewage and water samples from a bovine waste lagoon and a polluted creek. Amplification was observed in only one sheep sample out of 260 human and nonswine animal fecal samples. Sequencing of PCR products from pig feces demonstrated 100% similarity to pig mcrA sequence from clone P23-2. The minimal amount of DNA required for the detection was 1 pg for P23-2 plasmid, 1 ng for pig feces, 50 ng for swine waste lagoon surface water, 1 ng for sow waste influent, and 10 ng for lagoon sludge samples. Lower detection limits of 10(-6) g of wet pig feces in 500 ml of phosphate-buffered saline and 10(-4) g of lagoon waste in estuarine water were established for the P23-2 marker. This study was the first to utilize methanogens for the development of a swine-specific fecal contamination marker.     

Concentrations of Pathogens and Indicators in Animal Feces in the Sydney Watershed

A fecal analysis survey was undertaken to quantify animal inputs of pathogenic and indicator microorganisms in the temperate watersheds of Sydney, Australia. The feces from a range of domestic animals and wildlife were analyzed for the indicator bacteria fecal coliforms and Clostridium perfringens spores, the pathogenic protozoa Cryptosporidium and Giardia, and the enteric viruses adenovirus, enterovirus, and reovirus. Pathogen and fecal indicator concentrations were generally higher in domestic animal feces than in wildlife feces. Future studies to quantify potential pathogen risks in drinking-water watersheds should thus focus on quantifying pathogen loads from domestic animals and livestock rather than wildlife.     

Urinary and fecal immunoglobulin A, cortisol and 11-17 dioxoandrostanes, and serum cortisol in metabolic cage housed female cynomolgus monkeys (Macaca fascicularis)

BACKGROUND AND METHODS: Quantitative enzyme-immunoassays of urinary and fecal immunoglobulin A (IgA), cortisol and 11-17-dioxoandrostanes (11,17-DOA), and serum cortisol in eight metabolic-cage-housed female cynomolgus monkeys were performed. The monkeys were divided into two groups, B and NB. Group B animals were blood sampled every 6 hours, whereas Group NB animals were not handled/blood sampled. RESULTS: No differences were recorded between the amounts of feces and urine excreted by the two groups. Group B animals excreted more urinary cortisol than did Group NB animals indicating that restraint-blood sampling resulted in a stress response. Excreted amounts of IgA and 11,17-DOA (urine and feces) did not differ between the groups. CONCLUSIONS: Urinary cortisol was a reliable marker of the stress associated with repeated blood sampling. Declining amounts of excreted urinary cortisol indicated that cynomolgus monkeys acclimated quickly to repeated blood sampling in metabolism cages. Within and between animal variation in amounts of feces voided demonstrated the importance of expressing fecal markers as 'amounts excreted per time unit per kg body weight' rather than just measuring the concentrations in fecal samples.     

Impacts of natural history and exhibit factors on carnivore welfare

To improve the welfare of nonhuman animals under professional care, zoological institutions are continuously utilizing new methods to identify factors that lead to optimal welfare. Comparative methods have historically been used in the field of evolutionary biology but are increasingly being applied in the field of animal welfare. In the current study, data were obtained from direct behavioral observation and institutional records representing 80 individual animals from 34 different species of the order Carnivora. Data were examined to determine if a variety of natural history and animal management factors impacted the welfare of animals in zoological institutions. Output variables indicating welfare status included behavioral diversity, pacing, offspring production, and infant mortality. Results suggested that generalist species have higher behavioral diversity and offspring production in zoos compared with their specialist counterparts. In addition, increased minimum distance from the public decreased pacing and increased offspring production, while increased maximum distance from the public and large enclosure size decreased infant mortality. These results have implications for future exhibit design or renovation, as well as management practices and priorities for future research.     

Pregnancy diagnosis in zoo animals by estrogen determination in feces

Estrogen concentration in feces was investigated in five different herbivorous species of zoo animals. Using a nonspecific estrogen radioimmunoassay, in four species (red buffalo, yak, Grevy's zebra, and Nubian ibex) pregnancy was revealed by measuring estrogen concentration in feces. In hippopotamus, the levels of fecal estrogens were not different between pregnant and nonpregnant animals.     

Effect of felid activity on zoo visitor interest

The extent to which the presence of zoo visitors influences animal behavior, and the ways in which animal activity influences visitor interest and perception, are of great interest to zoological parks. Visitors have been variously characterized as being enriching for zoo animals, as being stressors, and generally as influencing behavior in measurable ways. Most studies have focused on primates, and have assumed a “visitor effect” paradigm (i.e., visitors influence animal behavior). Here we present findings from a study of a nonprimate group (felids), and examine the “visitor attraction” model, which assumes that visitors are attracted to active animals. We assessed visitor interest and number at seven cat exhibits at the Brookfield Zoo during the spring and summer of 2002. Data were collected during 1‐min scans of each exhibit at 10‐min intervals. The results indicate that visitor presence per se did not influence cat activity, and that visitor interest was generally greater when cats were active. Various species differences may be explained by visitor familiarity with the species, variations in exhibit design, and species‐specific activity budgets. We conclude that the visitor attraction model may be more appropriate for taxa, such as large cats, that tend naturally to be largely inactive and to respond little (if at all) to visitor disturbances or efforts to engage. The relationship must be viewed as bidirectional: visitors influence animal behavior, and animal behavior influences visitor interest. However, the strength and primary direction of this relationship is likely taxon‐specific. We suggest that a visitor attraction model may be more appropriate not only for felids, but for other taxa with similar behavioral patterns and responses as well. Zoo Biol 22:587–599, 2003. © 2003 Wiley‐Liss, Inc.     

Mosquito bloodmeal preferences in two zoological gardens in Germany

Because they provide a high density and diversity of vertebrate species, small water pools and shaded environments, zoological gardens offer ideal living conditions for numerous mosquito species. Depending on their host preferences and vector competencies, these species may be able to transmit pathogens between native and non‐adapted exotic blood host species, thereby causing morbidity and mortality among valuable zoo animals. To determine the extent to which native mosquito species feed on captive and wild animals, as well as on humans, in two German zoological gardens, mosquitoes were collected over two seasons by trapping and aspirating. A total of 405 blood‐fed specimens belonging to 16 mosquito taxa were collected. Genetic bloodmeal analysis revealed 56 host species, mainly representing mammals of the zoo animal population, including exotic species previously not known as blood hosts of the mosquito species collected. These results indicate opportunistic feeding patterns with low host‐specificity in the analysed mosquitoes, although these could be grouped, according to their bloodmeals, into ‘amphibian‐’, ‘non‐human mammal‐’ and ‘non‐human mammal and human‐’ feeding species. As the blood‐feeding preferences of vector‐competent mosquito species are major determinants of vector capacity, information on the blood‐feeding behaviour of mosquitoes in zoos is crucial to the success of targeted vector management.     

Development of a Swine-Specific Fecal Pollution Marker Based on Host Differences in Methanogen mcrA Genes

The goal of this study was to evaluate methanogen diversity in animal hosts to develop a swine-specific archaeal molecular marker for fecal source tracking in surface waters. Phylogenetic analysis of swine mcrA sequences compared to mcrA sequences from the feces of five animals (cow, deer, sheep, horse, and chicken) and sewage showed four distinct swine clusters, with three swine-specific clades. From this analysis, six sequences were chosen for molecular marker development and initial testing. Only one mcrA sequence (P23-2) showed specificity for swine and therefore was used for environmental testing. PCR primers for the P23-2 clone mcrA sequence were developed and evaluated for swine specificity. The P23-2 primers amplified products in P23-2 plasmid DNA (100%), pig feces (84%), and swine waste lagoon surface water samples (100%) but did not amplify a product in 47 bacterial and archaeal stock cultures and 477 environmental bacterial isolates and sewage and water samples from a bovine waste lagoon and a polluted creek. Amplification was observed in only one sheep sample out of 260 human and nonswine animal fecal samples. Sequencing of PCR products from pig feces demonstrated 100% similarity to pig mcrA sequence from clone P23-2. The minimal amount of DNA required for the detection was 1 pg for P23-2 plasmid, 1 ng for pig feces, 50 ng for swine waste lagoon surface water, 1 ng for sow waste influent, and 10 ng for lagoon sludge samples. Lower detection limits of 10⁻⁶ g of wet pig feces in 500 ml of phosphate-buffered saline and 10⁻⁴ g of lagoon waste in estuarine water were established for the P23-2 marker. This study was the first to utilize methanogens for the development of a swine-specific fecal contamination marker.     

A Systematic Review and Meta-Analysis of the Campylobacter spp. Prevalence and Concentration in Household Pets and Petting Zoo Animals for Use in Exposure Assessments

Animal contact is a potential transmission route for campylobacteriosis, and both domestic household pet and petting zoo exposures have been identified as potential sources of exposure. Research has typically focussed on the prevalence, concentration, and transmission of zoonoses from farm animals to humans, yet there are gaps in our understanding of these factors among animals in contact with the public who don’t live on or visit farms. This study aims to quantify, through a systematic review and meta-analysis, the prevalence and concentration of Campylobacter carriage in household pets and petting zoo animals. Four databases were accessed for the systematic review (PubMed, CAB direct, ProQuest, and Web of Science) for papers published in English from 1992–2012, and studies were included if they examined the animal population of interest, assessed prevalence or concentration with fecal, hair coat, oral, or urine exposure routes (although only articles that examined fecal routes were found), and if the research was based in Canada, USA, Europe, Australia, and New Zealand. Studies were reviewed for qualitative synthesis and meta-analysis by two reviewers, compiled into a database, and relevant studies were used to create a weighted mean prevalence value. There were insufficient data to run a meta-analysis of concentration values, a noted study limitation. The mean prevalence of Campylobacter in petting zoo animals is 6.5% based on 7 studies, and in household pets the mean is 24.7% based on 34 studies. Our estimated concentration values were: 7.65x10³cfu/g for petting zoo animals, and 2.9x10⁵cfu/g for household pets. These results indicate that Campylobacter prevalence and concentration are lower in petting zoo animals compared with household pets and that both of these animal sources have a lower prevalence compared with farm animals that do not come into contact with the public. There is a lack of studies on Campylobacter in petting zoos and/or fair animals in Canada and abroad. Within this literature, knowledge gaps were identified, and include: a lack of concentration data reported in the literature for Campylobacter spp. in animal feces, a distinction between ill and diarrheic pets in the reported studies, noted differences in shedding and concentrations for various subtypes of Campylobacter, and consistent reporting between studies.     

有蹄类食性研究方法及研究进展

国内外有蹄类食性的研究方法主要有直接观察法、利用法、胃分析法和粪便显微分析法。近年来 ,粪样 DNA分析技术逐渐运用到对动物食性的判定上 ,其中粪便显微分析法是近 2 0年来国内研究有蹄类食性的最主要方法。对这些方法的优势和局限性进行了评价 ,并简述了国内外有蹄类食性研究进展和发展趋势     

Occurrence of Rhodococcus coprophilus and associated actinomycetes in feces, sewage, and freshwater.

Freshwater, sewage, and fecal samples from various sources were examined for Rhodococcus coprophilus, associated actinomycetes, Escherichia coli, and fecal streptococci. Rhodococcus coprophilus was isolated consistently from feces of farm animals, poultry reared in proximity to farm animals, freshwater, and wastewater polluted with animal fecal wastes. It was not isolated from samples of human feces. The ratio of R. coprophilus total actinomycetes was higher in feces from cattle, sheep, ducks, and geese than in specimens from pigs, horses, and fowl. In samples from two freshwater streams polluted by fecal material from farm animals, the ratios of R. copropilus to total actinomycetes were similar to those found in fecal specimens from cattle and sheep. Ratios of fecal coliform to fecal streptococci could not distinguish between fresh human and animal fecal samples and, furthermore, were not reflected in the stream waters polluted by animal fecal material. R. coprophilus has potential in water and dairy bacteriology as a specific indicator organism of fecal pollution due to farm animal wastes.     

A versatile enzyme immunoassay for the determination of progestogens in feces and serum

The ability of zoos to monitor the reproductive status of their animals can vastly improve the effectiveness of husbandry/management practices, and noninvasive methods such as fecal steroid analysis are the easiest to apply in a zoo setting. Furthermore, enzyme immunoassay (EIA) is preferred to radioimmunoassay (RIA) as the method of quantifying hormones because EIAs do not involve the use, storage, and disposal of radioactive materials. However, progesterone is excreted in the feces as predominantly unconjugated metabolites (progestogens) and, until recently, antibodies able to cross‐react with a variety of progestogens were used primarily in RIAs. An EIA using a broad‐spectrum progestogen antibody is described and applied to serum and/or fecal samples from female African elephants, black rhinoceros, white rhinoceros, okapi, and hippopotami. The clear progestogen profiles generated in these species suggest that the described EIA would be as versatile as the RIA using the same antibody and could be a practical and economical alternative to RIAs for monitoring gonadal function via progestogen analysis in zoo species. Zoo Biol 20:227–236, 2001. © 2001 Wiley‐Liss, Inc.