Microbiology Commons^™

Group Size And Nest Spacing Affect Buggy Creek Virus (Togaviridae: Alphavirus) Infection In Nestling House Sparrows, Valerie A. Brown, Charles R. Brown

Papers in Ornithology

The transmission of parasites and pathogens among vertebrates often depends on host population size, host species diversity, and the extent of crowding among potential hosts, but little is known about how these variables apply to most vector-borne pathogens such as the arboviruses (arthropod-borne viruses). Buggy Creek virus (BCRV; Togaviridae: Alphavirus) is an RNA arbovirus transmitted by the swallow bug (Oeciacus vicarius) to the cliff swallow (Petrochelidon pyrrhonota) and the introduced house sparrow (Passer domesticus) that has recently invaded swallow nesting colonies. The virus has little impact on cliff swallows, but house sparrows are seriously …

Interspecies Comparison Of Αii-Spectrin Abundance Between Chinook Salmon And Steelhead, Brielle D. Kemis, Ann L. Miracle, Katie A. Wagner, Christa M. Woodley

STAR Program Research Presentations

Salmonids, such as Chinook salmon (Oncorhynchus tshawytscha) and steelhead (O. mykiss), are a staple economic, recreational, tribal, and environmental resource, yet many populations are unsustainable. This study was part of a broad scale effort to monitor the impact of downstream migration obstacles on juvenile salmonid health and survival, which is an essential step towards increasing Smolt-to-Adult Return ratios (SARs). The objective of this study was to determine if juvenile Chinook salmon and steelhead exhibit differing quantities of alphaII-Spectrin Breakdown Products (SBDPs) over two consecutive spring migration periods, indicative of neurogenesis rate and/or biological response to head …

Effect Of The Infection With Porcine Reproductive And Respiratory Syndrome Virus On The Regulation Of Cytokines: Tumor Necrosis Factor-Alpha And Interleukin-10, Sakthivel Subramaniam

School of Veterinary and Biomedical Sciences: Dissertations, Theses, and Student Research

Porcine reproductive and respiratory syndrome virus (PRRSV) causes late-term abortion in sows and pneumonia in growing piglets. PRRSV evades the host immune response by several mechanisms, including the modulation of cytokine secretions in infected pigs, which is the subject of this dissertation. Particularly, PRRSV reduces the secretion of the pro-inflammatory cytokine, tumor necrosis factor-α (TNF-α) but increases the secretion of the anti-inflammatory cytokine interleukin-10 (IL-10). The latter effect, however, is PRRSV strain-specific. In this dissertation, we have examined mechanisms by which PRRSV regulates TNF-α and IL-10 expressions. The pathogenic strain FL12, derived from a PRRSV infectious clone, consistently suppressed TNF-α …

Isolation By Distance Explains Genetic Structure Of Buggy Creek Virus, A Bird-Associated Arbovirus, Abinash Padhi, Amy T. Moore, Mary Bomberger Brown, Jerome E. Foster, Martin Pfeffer, Charles R. Brown

School of Natural Resources: Faculty Publications

Many of the arthropod-borne viruses (arboviruses) show extensive genetic variability and are widely distributed over large geographic areas. Understanding how virus genetic structure varies in space may yield insight into how these pathogens are adapted to and dispersed by different hosts or vectors, the relative importance of mutation, drift, or selection in generating genetic variability, and where and when epidemics or epizootics are most likely to occur. However, because most arboviruses tend to be sampled opportunistically and often cannot be isolated in large numbers at a given locale, surprisingly little is known about their spatial genetic structure on the local …

Western Australia's Biosecurity, Department Of Agriculture And Food, Western Australia, Department Of Environment And Conservation, Western Australia, Department Of Fisheries, Western Australia, Forest Products Commission, Western Australia

Biosecurity published reports

Biosecurity is the protection of the economy, environment and human health from the negative impacts of pests, weeds and diseases, including the management of invasive species if they become established (definition adopted by AusBIOSEC).

This requires steps to prevent the entry of harmful organisms, as well as the timely and efficient management of them if they do enter.

Biosecurity threats range from catastrophic diseases such as Foot and Mouth Disease, which causes rapid and widespread animal deaths, to more subtle and slower spreading problems such as invasive weeds, mussels that foul vessel hulls and fungus-induced dieback disease of trees. These …

Characterization Of Photochromogenic Mycobacterium Spp. From Chesapeake Bay Striped Bass Morone Saxatilis, D. T. Gauthier, A. M. Helenthal, M. W. Rhodes, W. K. Vogelbein, H. I. Kator

Biological Sciences Faculty Publications

A large diversity of Mycobacterium spp. has been isolated from striped bass Morone saxatilis in Chesapeake Bay, USA. The new species M. shottsii and M. pseudoshottsii are the dominant isolates, while the classical fish pathogen M. marinum is found much less frequently. M. fortuitum and M. chelonae, other Mycobacterium spp. known to commonly infect fishes, have not yet been aseptically isolated from striped bass within Chesapeake Bay. While M. pseudoshottsii and M. shottsii have been phenotypically and genotypically characterized, other less common mycobacterial isolates have not. In the present study, we describe 17 photochromogenic isolates from Chesapeake …

Ultraviolet Light As An Adjunct Treatment To Pasteurization For Microbial Reduction In Milk Intended For Extended Shelf Life, Jacob Zonneveld

Dairy Science

The objective of this study was to determine whether ultraviolet light treatment of milk could be used in adjunct with pasteurization and to determine whether the microbial count in bulk tank raw milk was sufficient enough to receive significant results. Samples were collected from a bulk tank filled with 1600 gallons of raw Holstein and Jersey milk from the Cal Poly Dairy throughout the course of a week. Control (library) samples were also taken from the raw milk tank and stored without air or agitation for comparison. These samples were plated each day and examined 24 and 48 hours after …

Winter Ecology Of Buggy Creek Virus (Togaviridae, Alphavirus) In The Central Great Plains, Charles R. Brown, Stephanie A. Strickler, Amy T. Moore, Sarah A. Knutie, Abinash Padhi, Mary Bomberger Brown, Ginger R. Young, Valerie A. O'Brien, Jerome E. Foster, Nicholas Komar

School of Natural Resources: Faculty Publications

A largely unanswered question in the study of arboviruses is the extent to which virus can overwinter in adult vectors during the cold winter months and resume the transmission cycle in summer. Buggy Creek virus (BCRV; Togaviridae, Alphavirus) is an unusual arbovirus that is vectored primarily by the swallow bug (Hemiptera: Cimicidae: Oeciacus vicarius) and amplified by the ectoparasitic bug’s main avian hosts, the migratory cliff swallow (Petrochelidon pyrrhonota) and resident house sparrow (Passer domesticus). Bugs are sedentary and overwinter in the swallows’ mud nests. We evaluated the prevalence of BCRV and extent of …

Ecology And Relationships Of Rhabdias Spp. (Nematoda: Rhabdiasidae) From North American Amphibians And Reptiles, Gabriel J. Langford

School of Biological Sciences: Dissertations, Theses, and Student Research

Lungworms of the cosmopolitan genus Rhabdias (Nematoda: Rhabdiasidae) are among the most common parasites of amphibians and squamate reptiles. The life cycles, life histories, host specificities, and evolutionary relationships of Rhabdias spp. were studied through examination of their free-living and parasitic stages in amphibians and reptiles. This study found free-living development of anuran lungworms was primarily limited to heterogonic reproduction, whereas snake lungworms primarily reproduced homogonically. Infective anuran lungworms penetrated the skin of frogs and toads; in contrast, snake lungworms penetrated snake esophageal tissue during per os infections. Our molecular phylogeny strongly supported separate clades for anuran and snake lungworms, …

Survival Of Mycoplasma Species In Recycled Bedding Sand And Possible Implications For Disease Transmission To Ruminants, Anne E. Justice-Allen

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Mycoplasmas are a group of bacteria which are small in size, lack a cell wall, and have small genomes in comparison to other bacteria. The members of the group that are pathogenic utilize several mechanisms to evade the host's immune system. These processes affect surveillance and control mechanisms such as serologic testing and vaccination. Many of these organisms cause diseases of livestock, which heavily impact production parameters such as weight gain, milk yield, and egg production. Mycoplasmas also cause disease in people.

Mycoplasma spp. can cause mastitis, metritis, pneumonia, and arthritis. The currently documented routes of transmission of Mycoplasma spp. …

Quantitative Pcr Assay For Mycobacterium Pseudoshottsii And Mycobacterium Shottsii And Application To Environmental Samples And Fishes From The Chesapeake Bay, D. T. Gauthier, K. S. Reece, J. Xiao, M. W. Rhodes, H. I. Kator, R. J. Latour, C. F. Bonzek, J. M. Hoenig, W. K. Vogelbein

Biological Sciences Faculty Publications

Striped bass (Morone saxatilis) in the Chesapeake Bay are currently experiencing a very high prevalence of mycobacteriosis associated with newly described Mycobacterium species, Mycobacterium pseudoshottsii and M. shottsii. The ecology of these mycobacteria outside the striped bass host is currently unknown. In this work, we developed quantitative real-time PCR assays for M. pseudoshottsii and M. shottsii and applied these assays to DNA extracts from Chesapeake Bay water and sediment samples, as well as to tissues from two dominant prey of striped bass, Atlantic menhaden (Brevoortia tyrannus) and bay anchovy (Anchoa mitchilli). Mycobacterium …

Effects Of The Pathogenic Water Mold Saprolegnia Ferax On Survival Of Amphibian Larvae, John M. Romansic, Kristin A. Diez, Elise M. Higashi, James E. Johnson, Andrew R. Blaustein

All Faculty Scholarship for the College of the Sciences

Infectious diseases are a significant threat to worldwide biodiversity. Amphibian declines, a significant part of current biodiversity losses, are in many cases associated with infectious disease. Water molds are one group of pathogens affecting amphibians on a worldwide basis. Although water molds have been studied extensively for their effects on host embryos, little information is available about how they affect post-embryonic amphibians. We tested the effects of one species of water mold, Saprolegnia ferax, in a comparative study of larvae of 4 amphibian species: Pseudacris regilla (Pacific treefrog), Rana cascadae (Cascades frog), Ambystoma macrodactylum (long-toed salamander), and R. aurora (red-legged …

Host And Seasonal Effects On The Infection Dynamics Of Skrjabinoptera Phrynosoma (Ortlepp) Schulz, 1927, A Parasitic Nematode Of Horned Lizards, Kathryn Claire Hilsinger

College of Graduate Studies: Theses & Dissertations

Author's Abstract: Skrjabinoptera phrynosoma (Ortlepp) Schulz, 1927 is a common parasitic nematode of horned lizards. The life cycle of S. phrynosoma was described by Lee in 1957, but has received little attention since. The present study addressed effect of season as well as host characteristics on the infection dynamics in lizard hosts. In the Alvord Basin in southeastern Oregon, S. phrynosoma were collected from Phrynosoma platyrhinos Gerard 1852 horned lizards via stomach flushes, cloaca flushes and fecal pellet collections. Parasite load variables (number of nematodes per host, length of those nematodes, and total worm burden (ΣL)) were analyzed within three …

Letter From The Dean, Lalit Verma

Discovery, The Student Journal of Dale Bumpers College of Agricultural, Food and Life Sciences

No abstract provided.

Peptide Hydrolysis And The Uptake Of Dipeptides By Phytoplankton, Margaret R. Mulholland, Cindy Lee

OES Faculty Publications

Rates of peptide hydrolysis (using the fluorescent substrate, lucifer yellow anhydride-labeled tetra-alanine) and dipeptide uptake (using dually labeled, ¹⁵N and ¹³C, dialanine) were measured in phytoplankton cultures and in natural populations during algal blooms dominated by one or two taxa. During most sampling events, both peptide hydrolysis and dipeptide uptake were greatest in the size fraction containing the dominant phytoplankter, suggesting that phytoplankton contribute substantially to or may even dominate observed extracellular peptide hydrolysis and dipeptide uptake in the environment. These are the first data suggesting that dipeptides may be taken up directly by phytoplankton and this may …

Phylogeographical Structure And Evolutionary History Of Two Buggy Creek Virus Lineages In The Western Great Plains Of North America, Abinash Padhi, Amy T. Moore, Mary Bomberger Brown, Jerome E. Foster, Martin Pfeffer, Kathryn P. Gaines, Valerie A. O'Brien, Stephanie A. Strickler, Allison E. Johnson, Charles R. Brown

School of Natural Resources: Faculty Publications

Buggy Creek virus (BCRV) is an unusual arbovirus within the western equine encephalitis complex of alphaviruses. Associated with cimicid swallow bugs (Oeciacus vicarius) as its vector and the cliff swallow (Petrochelidon pyrrhonota) and house sparrow (Passer domesticus) as its amplifying hosts, this virus is found primarily in the western Great Plains of North America at spatially discrete swallow nesting colonies. For 342 isolates collected in Oklahoma, Nebraska, Colorado, and North Dakota, from 1974 to 2007, we sequenced a 2076 bp region of the 26S subgenomic RNA structural glycoprotein coding region, and analyzed phylogenetic relationships, …

Experimental Inoculation Of House Sparrows (Passer Domesticus) With Buggy Creek Virus, Kathryn P. Huyvaert, Amy T. Moore, Nicholas A. Panella, Eric A. Edwards, Mary Bomberger Brown, Nicholas Komar, Charles R. Brown

School of Natural Resources: Faculty Publications

We performed experimental inoculations of house sparrows (Passer domesticus) with Buggy Creek virus (BCRV), a poorly known alphavirus (Togaviridae) vectored primarily by the swallow bug (Hemiptera: Cimicidae: Oeciacus vicarius) that is an ectoparasite of the cliff swallow (Petrochelidon pyrrhonota) and house sparrow. Viremias were detected by plaque assay in two of six birds on days 1–3 postinoculation; viremia was highest on day 2. Viral RNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in blood of six of 12 birds ranging from day 1 to day 15 postinoculation. Infectious BCRV was detected in …

Antimicrobial Activity Screening Of Recombinant And Synthetic Varasin A Defensin From The Hard Tick Dermacentor Variabilis Against Various Bacteria, Julia A. Sharp

Biological Sciences Theses & Dissertations

Hematophagous arthropods, such as ticks and mosquitoes, rely on their innate immune system for defense against pathogens ingested in a blood meal as well as those acquired through injury. In response to pathogen recognition, the production of antimicrobial peptides, such as defensin, is typically upregulated. Varisin, a defensin, is thought to be a key component in the immunocompetence of the hard tick D. variabilis against Borrelia burgdorferi, the causative agent of Lyme disease. To study the antimicrobial effects of varisin, recombinant varisin was expressed by both insect cells and E coli. Purification of the protein followed by enterokinase treatment yielded …

The Influence Of Early Experience On, And Inheritance Of, Cerebral Lateralization, Culum Brown, Jac Western, Victoria A. Braithwaite

Veterinary Science and Medicine Collection

Cerebral lateralization refers to the lateralized partitioning of cognitive function in either hemisphere of the brain. Using a standard detour test, we investigated lateralized behaviour in wild-caught, female poeciliid fish, Brachyraphis (=Brachyrhaphis) episcopi, from high- and low-predation areas. Wild fish were bred and their offspring reared under controlled laboratory conditions. These laboratory-reared fish were screened in the same laterality assays as their parents. We observed differences between wild-caught females and their laboratory-reared female offspring in the pattern of lateralization (tendency to use one hemisphere over the other to process information). Conversely, the strength of lateralization (consistency of hemispherical bias) was …

Detection Of Panulirus Argus Virus 1 In Caribbean Spiny Lobsters, Megan M. Montgomery-Fullerton, Roland Cooper, Kathryn M. Kauffman, Jeffrey D. Shields, Robert E. Ratzlaff

Collected Faculty Scholarship

Panulirus argus Virus 1 (PaV1) is a pathogenic virus that infects Caribbean spiny lobsters P. argus in the Florida Keys. We have developed a PCR detection assay for PaV1 for the purpose of studying the natural history of the virus and for monitoring the prevalence of infection. The detection of the virus in hemolymph and other tissues is based on the PCR amplification of a 499 bp product using specific primers designed from a cloned fragment of the PaV1 genome. The sensitivity limit for the assay was 1.2 fg of purified viral DNA. The PaV1 primers did not react with …

Characterization Of Dairy Milk House Waste Water In Kentucky, Anshu Singh, Czarena L. Crofcheck, Gail M. Brion

Biosystems and Agricultural Engineering Faculty Publications

This study focuses on characterization of milk house waste water from eight different farms in Kentucky. The farms were separated into three groups based on the number of cows: small (20-30), medium (30-60), and large (over 60 cows). Samples were collected once a month from four farms and twice a month from the remainder. Samples were analyzed for chemical, biochemical, and microbiological characteristics. Results indicated a large and significant variation in the chemical and microbiological characteristics between the farms. Farm size had a significant effect on the nutrient content of the waste water. Though samples exhibited seasonal variation, there was …

Bird Movement Predicts Buggy Creek Virus Infection In Insect Vectors, Charles R. Brown, Mary Bomberger Brown, Amy T. Moore, Nicholas Komar

School of Natural Resources: Faculty Publications

Predicting the spatial foci of zoonotic diseases is a major challenge for epidemiologists and disease ecologists. Migratory birds are often thought to be responsible for introducing some aviozoonotic pathogens such as West Nile and avian influenza viruses to a local area, but most information on how bird movement correlates with virus prevalence is anecdotal or indirect. We report that the prevalence of Buggy Creek virus (BCRV) infection in cimicid swallow bugs (Oeciacus vicarius), the principal invertebrate vector for this virus, was directly associated with the likelihood of movement by cliff swallows (Petrochelidon pyrrhonota), an amplifying host …

Ecological Correlates Of Buggy Creek Virus Infection In Oeciacus Vicarius, Southwestern Nebraska, 2004, Amy T. Moore, Eric A. Edwards, Mary Bomberger Brown, Nicholas Komar, Charles R. Brown

School of Natural Resources: Faculty Publications

Buggy Creek virus (family Togaviridae, genus Alphavirus, BCRV) is an alphavirus within the western equine encephalitis virus complex whose primary vector is the swallow bug, Oeciacus vicarius Horvath (Hemiptera: Cimicidae), an ectoparasite of the colonially nesting cliff swallow, Petrochelidon pyrrhonota, that is also a frequent host for the virus.We investigated ecological correlates of BCRV infection in 100-bug pools at 14 different swallow colony sites in southwestern Nebraska from summer 2004, by using plaque assay on Vero cells to identify cytopathic virus and reverse transcription-polymerase chain reaction to identify noncytopathic viral RNA. We found 26.7% of swallow bug pools …

Enzymes As Feed Additive To Aid In Responses Against Eimeria Species In Coccidia-Vaccinated Broilers Fed Corn-Soybean Meal Diets With Different Protein Levels, J. Parker, E. O. Oviedo Rondon, Beatrice A. Clack, S. Clemente-Hernandez, J. Osborne, J. C. Remus, H. Kettunen, H. Makivuokko, E. M. Pierson

Faculty Publications

This research aimed to evaluate the effects of adding a combination of exogenous enzymes to starter diets varying in protein content and fed to broilers vaccinated at day of hatch with live oocysts and then challenged with mixed Eimeria spp. Five hundred four 1-d-old male Cobb-500 chickens were distributed in 72 cages. The design consisted of 12 treatments. Three anticoccidial control programs [ionophore (IO), coccidian vaccine (COV), and coccidia-vaccine + enzymes (COV + EC)] were evaluated under 3 CP levels (19, 21, and 23%), and 3 unmedicated-uninfected (UU) negative controls were included for each one of the protein levels. All …

Properties Of Corneal Receptors In A Teleost Fish, Paul J. Ashley, Lynne U. Sneddon, Catherine R. Mccrohan

Veterinary Science and Medicine Collection

Corneal receptors have not previously been identified in lower vertebrates. The present study describes the properties of trigeminal ganglion corneal receptors in a teleost fish, the rainbow trout (Oncoryhnchus mykiss). Out of 27 receptors, 7 were polymodal nociceptors, 6 were mechanothermal nociceptors, 2 were mechanochemical receptors and the largest group, 12, were only responsive to mechanical stimulation. No cold responsive receptors were found on the trout cornea. Mechanical and thermal thresholds were lower and receptive field diameters smaller than those of cutaneous trigeminal receptors in the trout, demonstrating greater sensitivity in the cornea. The lack of cold sensitive neurons may …

Detection Of Panulirus Argus Virus 1 (Pav1) In The Caribbean Spiny Lobster Using Fluorescence In Situ Hybridization (Fish), Caiwen Li, Jeffrey D. Shields, Hamish J. Small, Kimberly S. Reece, Carmony L. Hartwig, Roland A. Cooper, Robert E. Ratzlaff

Collected Faculty Scholarship

Panulirus argus Virus 1 (PaV1) is the first virus known to be pathogenic to a wild lobster. It infects the Caribbean spiny lobster P. argus from the Florida Keys, and has a predilection for juveniles. The monitoring of the virus in wild populations and study of its behavior in the laboratory require the development of reliable diagnostic tools. A sensitive and specific fluorescence in situ hybridization (FISH) assay was developed for detection of PaV1. The lower detection limit using a 110 bp DNA probe in a dot-blot hybridization for PaV1 DNA was 10 pg of cloned template PaV1 DNA and …

Fisheries Research Contract Report No. 15 - Aquatic Animal Health Subprogram: Pilchard Herpesvirus Infection In Wild Pilchards (Frdc Project No. 2002/044) Final Report, Brian Jones, Melanie Crockford, Richard Whittington, Mark Crane, Graham Wilcox

Fisheries Research Contract Reports

In 1995 and again in 1998/9, millions of pilchards (Sardinops sagax neopilchardus) were found dead or dying off the coast of Australia. Some pilchards in New Zealand were also affected during the 1995 epizootic. The epizootics moved progressively in a "bushfire-like" manner against the prevailing currents at a rapid speed. An uncharacterised herpesvirus, pilchard herpesvirus (PHV), was associated with both mortality events and, based on its occurrence in affected fish and the disease pathogenesis, generally accepted as the causative agent, although experimental evidence of its role was not established. Until recently, rapid and sensitive methods to detect PHV were not …

Accumulation Of Phycotoxins In The Mussel Mytilus Galloprovincialis From The Central Adriatic Sea, Maja Pavela-Vrančić, Ivana Ujević, Živana Ninčević Gladan, Ambrose Furey

Physical Sciences Publications

Surveys of DSP (Diarrheic Shellfish Poisoning) toxin profiles in the mussel Mytilus galloprovincialis from coastal waters of the central Adriatic Sea, over the years 1995 to 2001, demonstrate that incidents of shellfish toxicity in this area are dominated by the occurrence of okadaic acid (OA) and a PTX derivative, 7-epi-pectenotoxin-2-seco acid (7-epi-PTX-2SA). Toxin composition and the relative ratio of toxic components in shellfish is in correlation with the occurrence of DSP producing organisms from the Dinophysis species along with Prorocentrum micans and Lingulodinium polyedrum. The occurrence of D. sacculus shortly before the appearance of OA in shellfish implicates its possible …

Defensin Transcript Distribution In Tissues And Life Stages Of The Ixodid Ticks Amblyomma Americanum And Ixodes Scapularis, Stephanie M. Todd

Biological Sciences Theses & Dissertations

A number of bacterial, viral, and protozoan diseases are transmitted to humans via arthropods. Arthropods possess an innate immune system to destroy invading microorganisms. However, arthropods unable to rid their systems of pathogens function as disease vectors. The American dog tick, Dermacentor variabilis, destroys invading Borrelia burgdorferi spirochetes, preventing transmission. In contrast, the black-legged tick, Ixodes scapularis, which is unable to destroy acquired B. burgdorferi, successfully vectors the pathogen. The Lone Star tick, Amblyomma americanum, vectors a similar pathogen called Borrelia lonestari. The distribution of defensin, an antimicrobial peptide of the innate immune system, was examined in A. americanum and/. …

Prevalence And Serovars Of Salmonella In The Feces Of Free-Ranging White-Tailed Deer (Odocoileus Virginianus) In Nebraska, David G. Renter, David P. Gnad, Jan M. Sargeant, Scott E. Hygnstrom

School of Natural Resources: Faculty Publications

To determine the prevalence and serovars of Salmonella in free-ranging deer, we cultured feces from white-tailed deer (Odocoileus virginianus) harvested by hunters during a regular firearm season in southeastern Nebraska (USA). We recovered Salmonella from 5 (1%; 95% confidence interval: 0.37– 2.20%) of 500 samples and identified four different Salmonella enterica serovars [Litchfield (1), Dessau (1), Infantis (2), and Enteritidis (1)]. Although the prevalence of Salmonella in free-ranging deer appears to be low, the serovars recovered are known to be pathogenic to humans and animals.