Characterization of Campylobacter jejuni, Campylobacter upsaliensis, and a novel Campylobacter sp. in a captive non-human primate zoological collection.

BACKGROUND: The aim of this study was to longitudinally investigate the prevalence and characterization of Campylobacter spp. from non-human primates primate (NHP) with a history of endemic diarrhea housed at Como Park Zoo. METHODS: Fecal samples from 33 symptom-free NHP belonging to eight different species were collected weekly for 9 weeks. Species-level characterization and phylogenetic analysis of isolates included biochemical testing and 16S rRNA sequencing. RESULTS: Campylobacter spp. were isolated from the feces of 42% (14/33) of the primates. Three Campylobacter spp. (C upsaliensis, C jejuni, and novel Campylobacter sp.) were identified from three NHP species. A possible positive host Campylobacter species-specificity was observed. However, no statistical association was observed between the isolation of Campylobacter spp. and age and sex of the animal. CONCLUSIONS: The study revealed the value of conducting repeated fecal sampling to establish the overall prevalence of Campylobacter in zoo-maintained NHP; it also importantly identifies a novel Campylobacter sp. isolated from white-faced saki monkeys.

Organic or antibiotic-free labeling does not impact the recovery of enteric pathogens and antimicrobial-resistant Escherichia coli from fresh retail chicken.

We investigated the implied health benefits of retail chicken breast labeled as "organic" or "antibiotic-free" when compared to conventional products based on frequency of contamination by Salmonella spp., Campylobacter spp., and coliform bacteria resistant to fluoroquinolones, extended-spectrum cephalosporins, or carbapenems. We purchased 231 prepackaged chicken breasts from 99 grocery stores representing 17 retail chains in Ohio, Michigan, and Pennsylvania from June to September 2012. Ninety-six (41.5%) packages were labeled "antibiotic free" and 40 (17.3%) were labeled "organic," with the remaining 95 (41.1%) making neither label claim. Salmonella were recovered from 56 (24.2%) packages, and the recovery rate was not different between product types. Five percent of packages contained Salmonella carrying the extended-spectrum cephalosporin resistance gene bla(CMY-2), representing 21.4% of Salmonella isolates. Campylobacter spp. were recovered from 10.8% of packages, with observed recovery rates similar for the three product types. Using selective media, we recovered Escherichia coli harboring bla(CMY-2) from over half (53.7%) of packages, with similar recovery rates for all product types. In addition, we recovered E. coli carrying bla(CTX-M) from 6.9% of packages, and E. coli with QRDR mutations from 8.2% of packages. Fluoroquinolone-resistant E. coli recovered using selective media were more common (p<0.05) in conventional (18.9%) compared to organic (0) and antibiotic-free (2.1%) packages. Our results indicate that, regardless of product type, fresh retail chicken breast is commonly contaminated with enteric pathogens associated with foodborne illness and commensal bacteria harboring genes conferring resistance to critically important antimicrobial drugs.

Changes in plasma testosterone levels and brain AVT cell number during the breeding season in the green treefrog.

We exposed groups of adult male green treefrogs, Hyla cinerea, to acoustic stimuli (natural chorus or random tones) for seven consecutive nights at three time points during their natural breeding season (May, July, and September) and assessed seasonal changes in plasma androgen levels and number of arginine vasotocin (AVT) immunoreactive cells in the brain over this time period. We also tested whether social cues altered either androgens or AVT-ir cell number or size at each time point. Finally, we analyzed how these factors related to calling behavior. Data were collected over two breeding seasons. Call rate (calls/h) was assessed during the stimulus time (i.e. 'evoked calling') and during the remainder of the day ('spontaneous calling'). Plasma hormone levels were measured at the end of the acoustic treatment when brains were collected for immunocytochemistry. Circulating androgen levels declined over the breeding season. Males exposed to chorus sounds, however, had higher androgen levels than males exposed to tones. AVT-ir cell number increased across the breeding season in the nucleus accumbens but not the amygdala, anterior preoptic area, or magnocellular preoptic area, and soma size decreased in the nucleus accumbens as cell number increased. Social stimulation had no significant influence on either AVT-ir cell measure. Evoked call rate was higher in males exposed to natural chorus sounds compared to those exposed to random tones, but did not change during the season. In contrast, spontaneous call rate was higher at the beginning of the breeding season compared to the end, and unlike evoked calling was correlated with circulating androgen levels across all treatments and time points. AVT-ir soma size was positively correlated with both evoked and spontaneous calling. These results suggest that social exposure can prolong the elevation of gonadal hormones in the bloodstream, thus mitigating or slowing the seasonal decline of such hormones. In contrast, social exposure does not affect the seasonal pattern of AVT-ir cell number or soma size. The reciprocal relationship between social cues and hormones and the subsequent effect on behavior may provide hidden benefits to animals engaging in social interactions. However, unlike steroid hormone levels, the seasonal change in AVT-ir cell number and size is not counteracted by social stimulation.

Campylobacter taeniopygiae sp. nov., Campylobacter aviculae sp. nov., and Campylobacter estrildidarum sp. nov., Novel Species Isolated from Laboratory-Maintained Zebra Finches.

Zebra finches (Taeniopygia guttata) are laboratory animal species commonly used for modeling neurobiology and learning. Historically, using bacterial culture, biochemical analysis, and 16S ribosomal RNA gene sequencing, bacterial isolates from feces of finches housed at Massachusetts Institute of Technology had been presumptively diagnosed as Campylobacter jejuni, which is commonly isolated from both domestic and wild birds. Although the zebra finches were not clinically affected, C. jejuni is a known zoonotic pathogen that causes gastroenteritis in humans worldwide. Human transmission is predominantly foodborne and associated with the consumption of contaminated poultry; however, humans can also become infected from contact with C. jejuni-infected reservoir hosts. Because C. jejuni-infected finches pose a risk to research personnel, a study was undertaken to investigate the prevalence and taxonomic identification of Campylobacter spp. present in the finch colony. Campylobacter spp. were isolated from a total of 26 finch fecal samples collected in 2003, 2010, and 2017. 16S ribosomal RNA sequencing of all isolates determined that they shared 99% identity with either C. jejuni or Campylobacter lari. Sixteen of the isolates were subjected to further biochemical characterization and atpA and rpoB gene sequence analysis. Based on these analyses, three clusters of Campylobacter species were identified. The draft whole-genome sequences were determined for one representative isolate from each cluster. A pan-genomic phylogenetic tree, average nucleotide identity, digital DNA-DNA hybridization, and orthologous gene analyses indicated that each isolate was its own novel species, distinct from C. jejuni and other avian Campylobacter species. We have named these novel species Campylobacter taeniopygiae, Campylobacter aviculae, and Campylobacter estrildidarum, and in each novel species, we identified virulence genes suggesting their pathogenic and zoonotic potential.

Sex and seasonal differences in morphology of limbic forebrain nuclei in the green anole lizard.

Sex and seasonal differences in the brain occur in many species and are often related to behavioral expression. For example, morphology of limbic regions involved in male sex behavior are larger in males than in females, and sometimes are larger in the breeding than non-breeding season. Morphology can often be altered in adulthood by manipulating levels of steroid hormones. In untreated green anole lizards, previous work indicated that neuron soma size and density did not differ between the sexes in the preoptic area (POA) or ventromedial nucleus of the amygdala (AMY), two brain regions involved in the control of male reproductive behaviors [O'Bryant, E.L., Wade, J., 2002. Seasonal and sexual dimorphisms in the green anole forebrain. Horm. Behav. 41, 384-395.]. However, soma size was larger in both areas in breeding than non-breeding animals. The current study examined sex and seasonal differences in estimated brain region volume and total neuron number in the POA, AMY, and the ventromedial hypothalamus (VMH), a region typically involved in female reproductive behaviors. The volume of the POA was larger in males, and the POA and VMH of breeding animals were larger than those of non-breeding individuals. Differences in cell number did not exist in either of these two regions. In contrast, neuron counts in the AMY were greater in non-breeding than breeding animals, but the volume did not differ between the seasons. These data suggest that the structure of limbic brain regions is dynamic in adulthood and that parallels between morphology and the expression of masculine behavior exist for the POA, whereas other relationships are more complicated.

Androgen receptor immunoreactivity in skeletal muscle: enrichment at the neuromuscular junction.

Potential cellular targets of androgen action within skeletal muscle of the rat were determined by comparing the cellular distribution of androgen receptor (AR)-positive nuclei in the highly androgen-responsive levator ani (LA) muscle with that of the relatively androgen-unresponsive extensor digitorum longus (EDL) muscle. We found that androgen responsiveness correlates with AR expression in muscle fibers and not in fibroblasts. Results indicate that a much higher percentage of myonuclei in the LA are AR(+) than in the EDL (74% vs. 7%), correlating with differences in androgen responsiveness. Both muscles contain an equivalent proportion of AR(+) fibroblasts (approximately 62%). AR(+) nuclei were not observed in terminal Schwann cells in either muscle. These results suggest that ARs within LA muscle fibers mediate the androgen-dependent survival and growth of the LA muscle and its motoneurons. We also observed an unexpected enrichment of AR(+) myonuclei and fibroblasts proximate to neuromuscular junctions, suggesting that ARs at muscle synapses may selectively regulate synapse-specific genes important for the survival and growth of motoneurons. Although castration reduced the proportion of AR(+) fibroblasts in both muscles, the proportion of AR(+) myonuclei was reduced only in the LA. As expected, testosterone treatment prevented these effects of castration but, unexpectedly, increased the proportion of AR(+) myonuclei in the EDL to above normal. These results suggest that how AR expression in skeletal muscle is influenced by androgens depends not only on the particular muscle but on the particular cell type within that muscle.

Current research in amphibians: studies integrating endocrinology, behavior, and neurobiology.

Amphibian behavioral endocrinology has focused on reproductive social behavior and communication in frogs and newts. Androgens and estrogens are critical for the expression of male and female behavior, respectively, and their effects are relatively clear. Corticosteroids have significant modulatory effects on the behavior of both sexes, as does the peptide neuromodulator arginine vasotocin in males, but their effects and interactions with gonadal steroids are often complex and difficult to understand. Recent work has shown that the gonadal hormones and social behavior are mutually reinforcing: engaging in social interactions increases hormone levels just as increasing hormone levels change behavior. The reciprocal interactions of hormones and behavior, as well as the complex interactions among gonadal steroids, adrenal steroids, and peptide hormones have implications for the maintenance and evolution of natural social behavior, and suggest that a deeper understanding of both endocrine mechanisms and social behavior would arise from field studies or other approaches that combine behavioral endocrinology with behavioral ecology.

Expression of nuclear receptor coactivators in androgen-responsive and -unresponsive motoneurons.

Adult rat lumbar motoneurons in the spinal nucleus of the bulbocavernosus (SNB) respond to androgens with an increase in soma size. This response is mediated by the androgen receptor (AR) in these motoneurons. Interestingly, other lumbar motoneurons in the rat possess the AR, yet do not respond to androgens in this fashion. This paradox suggests the existence and participation of nuclear receptor coregulators in conferring direct androgen-responsiveness to select motoneurons in the adult rat spinal cord. Nuclear receptor coregulators have received much attention recently for their proposed role in enhancing or repressing the transcriptional activity of steroid hormone receptors. The present study used immunocytochemistry to identify a number of nuclear receptor coactivators that are expressed by adult lumbar motoneurons: SRC-1, SRC-2, CBP, p300, and cJUN. Results of this study indicate that all five of these coactivators are abundantly expressed in the androgen-responsive SNB, and in two adjacent motor pools, the androgen-responsive dorsolateral nucleus (DLN), and the androgen-unresponsive retrodorsolateral nucleus (RDLN). While we detected significant regional differences for only SRC-1 and cJUN, the SNB consistently contained the highest percentage of immunoreactive motoneurons for all five cofactors examined. Our results indicate five different putative cofactors have the potential to participate in motoneuronal responses to androgens, since their distribution overlaps well with the distribution of ARs in these motoneurons.

Seasonal and sexual dimorphisms in the green anole forebrain.

During the breeding season, male green anole lizards extend a throat fan (dewlap) in courtship. This behavior is facilitated by testosterone (T). Females extend a much smaller dewlap less often, even with the same dose of T. During the nonbreeding season when T is low, dewlap extension is reduced. To determine if parallels exist between structure and function, we investigated neuron soma size and density in the preoptic area (POA) and ventromedial nucleus of the amygdala (AMY), which are involved in the display behavior, in breeding and nonbreeding males and females. Cells from breeding animals were larger than cells from nonbreeding animals, but they were not sexually dimorphic. No significant effects existed in neuron density. This experiment indicates that portions of the anole forebrain important for sexual behavior are plastic and might be influenced by seasonal changes in steroid hormones. To investigate whether T can reverse the seasonal difference in soma size in both sexes, gonadectomized nonbreeding anoles were implanted with an empty or T propionate-filled capsule; animals were also tested for male-typical courtship behavior. Males and females treated with T had higher rates of dewlap extension, but across treatment groups these rates were greater in males. Neuron soma size in the POA and AMY was larger in males than females, but no effects of treatment were detected. Taken together, the results indicate that T can stimulate behavior in the nonbreeding season and suggest that a dissociation exists between the regulation of the courtship display and soma size of relevant brain regions.

Sexual dimorphism in neuromuscular junction size on a muscle used in courtship by green anole lizards.

The green anole lizard exhibits seasonal courtship behavior that is sexually dimorphic. This courtship consists of the extension of a bright red throat fan (dewlap) associated with head-bobbing display behavior. While males extend their dewlaps in aggressive encounters as well as in courtship, females use their considerably smaller dewlaps much less frequently and mainly in agonistic encounters. In parallel, a number of components of the neuromuscular system controlling dewlap extension are greater in males than in females during the breeding season, including dewlap motoneuron soma size and muscle fiber size and number. These features do not seem to change substantially in adulthood, despite a dramatic decline in dewlap use during the nonbreeding season. We explored the morphology of this neuromuscular system in more detail in the present experiment in males and females during both the breeding and nonbreeding seasons. Fiber and whole muscle length (approximately perpendicular to the fibers) were measured. Acetylcholinesterase histochemistry was used to visualize neuromuscular junctions (NMJs), and the surface area and density of NMJs were assessed for each animal. During the breeding season, NMJ size was larger in males than in females, but NMJ density along each fiber was equivalent between the sexes. In addition, whole muscle length and that of individual muscle fibers, was larger in males than in females. However, when corrected for body size, the sex difference in muscle fiber length disappeared. In the nonbreeding season, the sexual dimorphisms were maintained, suggesting that these features do not change substantially due to differences in circulating testosterone or a difference in use across seasons. Overall, these results are consistent with the idea that enhanced NMJ size is a relatively stable feature of the dewlap muscle in adulthood that either facilitates or is a consequence of using a larger muscle to extend a bigger dewlap in males compared to females.