Differences in pathogenicity, response to vaccination, and innate immune responses in different types of ducks infected with a virulent H5N1 highly pathogenic avian influenza virus from Vietnam.

In a previous study, we found clear differences in pathogenicity and response to vaccination against H5N1 highly pathogenic avian influenza (HPAI; HA dade 2.3.4) between Pekin (Anas platyrhynchos var. domestica) and Muscovy (Cairina moschata) ducks vaccinated using a commercial inactivated vaccine (Re-1). The objective of the present study was to further investigate the pathogenicity of H5N1 HPAI viruses in different species of ducks by examining clinical signs and innate immune responses to infection with a different strain of H5N1 HPAI virus (HA clade 1) in two domestic ducks, Pekin and Muscovy, and one wild-type duck, mallard (Anas platyrhynchos). Protection conferred by vaccination using the Re-1 vaccine against infection with this virus was also compared between Pekin and Muscovy ducks. Differences in pathogenicity were observed among the virus-infected ducks, as the Muscovy ducks died 2 days earlier than did the Pekin and mallard ducks, and they presented more-severe neurologic signs. Conversely, the Pekin and mallard ducks had significantly higher body temperatures at 2 days postinfection (dpi) than did the Muscovy ducks, indicating possible differences in innate immune responses. However, similar expression of innate immune-related genes was found in the spleens of virus-infected ducks at this time point. In all three duck species, there was up-regulation of IFN-alpha, IFN-gamma, IL-6, CCL19, RIG-I, and MHC class I and down-regulation of MHC class II, but variable expression of IL-18 and TLR7. As in our previous study, vaccinated Muscovy ducks showed less protection against virus infection than did Pekin ducks, as evidenced by the higher mortality and higher number of Muscovy ducks shedding virus when compared to Pekin ducks. In conclusion, infection with an H5N1 HPAI virus produced a systemic infection with high mortality in all three duck species; however, the disease was more severe in Muscovy ducks, which also had a poor response to vaccination. The differences in response to virus infection could not be explained by differences in the innate immune responses between the different types of ducks when examined at 2 days dpi, and earlier time points need to be evaluated.

Evaluation of the effects of altitude on biological signatures of inflammation and anxiety- and depressive-like behavioral responses.

Over sixteen million people suffer from a depressive episode annually in the United States, with females affected at twice the rate of males. Little is known about the effects of exposure to high altitude on the risk of development of major depressive disorder, despite reports of higher suicide rates at higher altitudes. We hypothesize that exposure to hypobaric hypoxia at high altitude increases endophenotypes of self-directed suicidal violence, including biological signatures of chronic inflammation and vulnerability to anxiety-like and depressive-like behavioral responses in a sex-specific manner. Biological signatures of inflammation, including granulocyte:lymphocyte ratios, monocyte cell counts, and monocyte:lymphocyte ratios were assessed using complete blood count data, anhedonia, and anxiety- and depressive-like behavioral responses were evaluated. We assessed biological signatures of inflammation and behavioral responses in the open-field test, sucrose preference test, and modified Porsolt forced swim test in young adult male and female Long-Evans and Sprague Dawley rats. All tests were conducted near sea level (374 ft [114 m] elevation) and at moderate-high altitude (5430 ft [1655 m] elevation) during acclimation periods of one, two, three, four, and five weeks following shipment from a sea level animal breeding facility (N = 320, n = 8 per group). Exposure to moderate-high altitude induced a biological signature of increased inflammation, as evidenced by main effects of altitude for: 1) increased granulocyte:lymphocyte ratio; 2) increased count and relative abundance of circulating monocytes; and 3) increased monocyte:lymphocyte ratios. Exposure to moderate-high altitude also increased anhedonia as assessed in the sucrose preference test in both male and female rats, when data were collapsed across strain and time. Among male and female Long Evans rats, exposure to moderate-high altitude increased immobility in the forced swim test, without changing anxiety-like behaviors in the open-field test. Finally, granulocyte:lymphocyte ratios were correlated with anhedonia in the sucrose preference test. These data are consistent with the hypothesis that hypobaric hypoxia at moderate-high altitude induces persistent endophenotypes of self-directed suicidal violence including biological signatures of inflammation, anhedonia, and depressive-like behavioral responses.

Effects of Pelleting, Irradiation, and Autoclaving of Rodent Feed on MPV and MNV Infectivity.

Murine norovirus (MNV) and mouse parvovirus (MPV) are among the most common adventitial viruses seen in laboratory mice, and infections arise in barrier facilities despite rigorous biosecurity programs. Some authors have implicated nonsterilized feed as a source of MPV in rodent facilities, but none have conclusively documented viral particles in the feed. In this study, we hypothesized that both viruses can resist the pelleting process but not subsequent irradiation or autoclaving, thus revealing a potential source of outbreaks in rodent facilities. To test this hypothesis, we contaminated powdered feed with 10-fold concentrations of MNV and MPV and fed it to both Swiss Webster (SW) and C57BL/6NTac (B6) mice to determine a 'powdered ID50' according to seroconversion over a 28-d period. We repeated the experiment by using powdered feed that we contaminated with increasing viral doses (as no. of powdered ID50) and subsequently pelleted; from these results, we determined a 'pelleted ID50.' Finally we assessed the effect of irradiation and autoclaving on contaminated pellets by using the same experimental design. The powdered ID50 was relatively low and identical in both mouse strains (2.51 × 10² pfu) for MNV but higher in B6 (copy number, 3.20 × 106) than SW (3.98 × 104 copies) for MPV. As hypothesized, mice were infected by contaminated rodent feed despite the pelleting process. Indeed, pelleting resulted in a 1- to 2-log increase in ID50 in both strains for MNV and MPV. Irradiation and autoclaving of infected pellets effectively prevented seroconversion of mice exposed to all doses of MNV, whereas a single mouse seroconverted at the highest dose of MPV (1.35 × 107 copies). These data suggest that both MNV and MPV remain infectious after conditions reproducing the rodent chow pelleting process and that nonsterilized rodent chow might be a source of viral outbreaks.

Hypercortisolemia and Depressive-like Behaviors in a Rhesus Macaque (Macaca mulatta) Involved in Visual Research.

A 10-y-old cranially implanted rhesus macaque (Macaca mulatta) involved in visual research was presented for dull mentation and weight loss. Physical examination revealed alopecia and poor body conditioning, and bloodwork revealed marked hypercortisolemia (23 µg/dL). Differential diagnoses for hypercortisolemia, weight loss, and alopecia included Cushing and pseudo-Cushing syndromes. To further evaluate hypercortisolemia, we compared the urine cortisol:creatinine ratio (UCCR) at baseline and after low-dose dexamethasone suppression (LDDS) testing in the presenting animal and healthy naïve and implanted working monkeys. At baseline, UCCR was 10 times higher in the presenting macaque (118.1 ± 7.1) than in naïve animals (12.5 ± 12.8) and 3 times higher than in healthy implanted working macaques (44.4 ± 6.9); however, levels were suppressed similarly by dexamethasone in both the presenting animal and healthy controls. In addition, healthy implanted working macaques had significantly higher baseline UCCR levels than naïve controls, suggesting chronic stress in working animals. Abdominal ultrasonography and radiographs of the presenting animal revealed marked bilateral adrenal mineralization but no overt adrenal tumor or hyperplasia. Overall, these results excluded endogenous Cushing syndrome and prompted us to evaluate different causes of pseudo-Cushing syndrome, including depression. Using videorecordings to evaluate behavior, we used published criteria for macaque models of depression models, including huddling, to make a presumptive diagnosis of depression. The macaque was treated with fluoxetine (2 mg/kg PO daily), provided increased environmental enrichment, and followed over time by regular UCCR assessment and videorecordings. The animal improved clinically and behaviorally, and UCCR returned to levels observed in working implanted macaques (44.4) after 8 wk of treatment. This case highlights the potential effect of research-related work on stress and pathologic behaviors in macaques and demonstrates the utility of UCCR and LDDS for screening behavioral and hypothalamic-pituitary-adrenal abnormalities in these animals.

A "Pedi" Cures All: Toenail Trimming and the Treatment of Ulcerative Dermatitis in Mice.

Ulcerative Dermatitis (UD) is the most common cause of unplanned euthanasia in mice used in research, with prevalence rates reported between 4 and 21%. UD is characterized by a deep, ulcerative lesion that appears most commonly over the dorsal neck and is attendant with an intense pruritus. The underlying cause of UD is currently unknown, and as a consequence, there are no directed therapies that resolve lesions reliably. However, there is a growing body of evidence that suggests a behavioral component to the onset, maintenance, and progression of UD lesions. Scratching behavior in response to the intense pruritus associated with UD lesions may be an effective target for interventional therapies. We hypothesized that interfering with scratching behavior by trimming the toenails of mice with UD, would resolve UD lesions. To test this hypothesis, we first evaluated the efficacy of toenail trims with a single application of Vetericyn at the time of treatment versus our previous standard of care, topical Tresaderm applied daily. We found that toenail trims were significantly more effective at resolving lesions (n = 39 toenail trims, n = 100 Tresaderm, p<0.0001) with 93.3% of animals healing by 14 days (median time to lesion resolution). Furthermore, dorsal neck lesions did not recur by 42 days after a single toenail trim (n = 54); however, flank lesions did not resolve and the outcome of the two lesion distributions following treatment were significantly different (p<0.0001). Finally, we implemented toenail trims at an institutional level and found similar efficacies (approximately 90%) for toenail trims regardless of one-time topical supplement used (triple antibiotic ointment, Tresaderm, and Vetericyn, n = 55, 58, 18, p = 0.63). This is the first report of a highly effective treatment for one of the most serious welfare issues in laboratory mice.

Postoperative Analgesia Due to Sustained-Release Buprenorphine, Sustained-Release Meloxicam, and Carprofen Gel in a Model of Incisional Pain in Rats (Rattus norvegicus).

Postoperative analgesia in laboratory rats is complicated by the frequent handling associated with common analgesic dosing requirements. Here, we evaluated sustained-release buprenorphine (Bup-SR), sustained-release meloxicam (Melox-SR), and carprofen gel (CG) as refinements for postoperative analgesia. The aim of this study was to investigate whether postoperative administration of Bup-SR, Melox-SR, or CG effectively controls behavioral mechanical and thermal hypersensitivity in a rat model of incisional pain. Rats were randomly assigned to 1 of 5 treatment groups: saline, 1 mL/kg SC BID; buprenorphine HCl (Bup HCl), 0.05 mg/kg SC BID; Bup-SR, 1.2 mg/kg SC once; Melox-SR, 4 mg/kg SC once; and CG, 2 oz PO daily. Mechanical and thermal hypersensitivity were tested daily from day-1 through 4. Bup HCl and Bup-SR attenuated mechanical and thermal hypersensitivity on days 1 through 4. Melox-SR and CG attenuated mechanical hypersensitivity-but not thermal hypersensitivity-on days 1 through 4. Plasma concentrations, measured by using UPLC with mass spectrometry, were consistent between both buprenorphine formulations. Gross pathologic examination revealed no signs of toxicity in any group. These findings suggest that postoperative administration of Bup HCl and Bup-SR-but not Melox-SR or CG-effectively attenuates mechanical and thermal hypersensitivity in a rat model of incisional pain.

Pekin and Muscovy ducks respond differently to vaccination with a H5N1 highly pathogenic avian influenza (HPAI) commercial inactivated vaccine.

Domestic ducks are key intermediates in the transmission of H5N1 highly pathogenic avian influenza (HPAI) viruses, and therefore are included in vaccination programs to control H5N1 HPAI. Although vaccination has proven effective in protecting ducks against disease, different species of domestic ducks appear to respond differently to vaccination, and shedding of the virus may still occur in clinically healthy vaccinated populations. In this study we compared the response to vaccination between two common domestic duck species, Pekin (Anas platyrhynchos domesticus) and Muscovy (Cairina moschata), which were vaccinated with a commercial inactivated vaccine using one of three different schedules in order to elicit protection to H5N1 HPAI before one month of age. Clear differences in responses to vaccination were observed; the Muscovy ducks developed lower viral antibody titers induced by the same vaccination as Pekin ducks and presented with higher morbidity and mortality after challenge with an H5N1 HPAI virus. When comparing the response to infection in non-vaccinated ducks, differences were also observed, with infected Muscovy ducks presenting a lower mean death time and more severe neurological signs than Pekin ducks. However Pekin ducks had significantly higher body temperatures and higher levels of nitric oxide in the blood at 2 days post challenge than Muscovy ducks, indicating possible differences in innate immune responses. Comparison of the expression of innate immune related genes in spleens of the non-vaccinated infected ducks showed differences including significantly higher levels of expression of RIG-I in Pekin ducks and of IL-6 in Muscovy ducks. Both duck species showed an up-regulation of IFNα and MHC-I expression, and a down-regulation of MHC-II. In conclusion, differences in response to infection and vaccination were observed between the two domestic duck species. This information should be taken into account when developing effective vaccination programs for controlling H5N1 HPAI in different species of ducks.

Adaptation and transmission of a duck-origin avian influenza virus in poultry species.

A duck-origin avian influenza virus (AIV) was used to study viral adaptation and transmission patterns in chickens (Gallus gallus domesticus) and Pekin ducks (Anas platyrhynchos domesticus). Inoculated birds were housed with naïve birds of the same species and all birds were monitored for infection. The inoculating duck virus was transmitted effectively by contact in both species. Viruses recovered from infected birds showed mutations as early as 1 or 3 days after inoculation in chickens and ducks, respectively. Amino acid substitutions in hemagglutinin (HA) or deletions in neuraminidase (NA) stalk regions were identified in chicken isolates, but only substitutions in HA were identified in duck isolates. HA substitution-containing viruses replicated more efficiently than those with NA stalk deletions. NA deletion mutants were not recovered from contact chickens, suggesting inefficient transmission. Amino acid substitutions in HA proteins appeared in pairs in chickens, but were independent in ducks, indicating adaptation in chickens. In addition, our findings showed that a duck-origin virus can rapidly adapt to chickens, suggesting that the emergence of new epidemic AIV can be rapid.

Immune-related gene expression in response to H11N9 low pathogenic avian influenza virus infection in chicken and Pekin duck peripheral blood mononuclear cells.

The duck and chicken are important hosts of avian influenza virus (AIV) with distinctive responses to infection. Frequently, AIV infections in ducks are asymptomatic and long-lasting in contrast to the clinically apparent and transient infections observed in chickens. These differences may be due in part to the host response to AIV infection. Using real-time quantitative PCR, we examined the expression of immune-related genes in response to low pathogenic AIV H11N9 infection in peripheral blood mononuclear cells (PBMC) isolated from the blood of chickens and Pekin ducks. While chicken PBMC expressed IL-1beta and IL-6 at high levels similar to mammalian species, duck PBMC expression levels were minimal or unchanged. Similarly, duck IFN-beta expression was nearly unaffected, whereas chicken expression was highly upregulated. Chicken IFN-gamma was expressed to higher levels than duck IFN-gamma, while IFN-alpha was expressed similarly by both species. IL-2 was elevated early in infection in duck PBMC, but returned to baseline levels by the end of the experiment; in contrast, IL-2 was weakly induced in chicken PBMC at late time points. TLR-7 and MHC class I molecule expressions were conserved between species, whereas duck MHC class II expression was downregulated and chicken expression was unchanged. These results show distinct PBMC expression patterns of pro-inflammatory cytokines and IFNs between species. The differences in pro-inflammatory cytokine and IFN expression reflect the asymptomatic and lasting infection observed in ducks and the tendency towards clinical signs and rapid clearance seen in chickens. These results highlight important differences in the host response to AIV of two species thought to be critical in the genesis and maintenance of epidemic strains of AIV.