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A mortality event of nine threatened smoky mice (Pseudomys fumeus) occurred in January 2020 at a captive breeding facility in southeastern Australia that was affected at the time by hazardous levels of bushfire smoke, despite being more than 20 km from the nearest fire. Pathologic and clinical observations indicated smoke inhalation was the cause of death. All animals had significant pulmonary lesions, notably pulmonary edema and congestion, and moderate amounts of dark brown to black pigmented intracellular and extracellular particles from <0.5-2.5 µm in diameter were observed in the central or hilar region of the lungs of four of six animals examined histologically. Deaths occurred between three and 30 d after exposure to smoke and, for seven animals in outdoor acclimatization enclosures, were associated with very high ambient temperature (>40 C). Similar mortalities did not occur in co-located parrots, suggesting differing species sensitivity to smoke inhalation. Our findings highlight the potential for smoke to be an underdiagnosed cause of mortality in free-ranging wildlife during bushfires and for bushfires to affect wildlife populations outside of burnt areas, including in unburnt refugia. Conservation interventions for wildlife after bushfires should consider and, where possible, mitigate the risk of animals dying due to increased respiratory demand following smoke inhalation injury., (© Wildlife Disease Association 2021.)

Causes of morbidity and mortality and a survey of infectious disease agents were collated from wild and colony-raised endangered Amargosa voles ( Microtus californicus scirpensis ). Six voles from the wild and 295 voles in the captive-breeding colony were included in the study upon identification of an infectious agent during screening, identification of clinical signs of disease, or finding a pathological condition or infectious agent on necropsy. Findings included 28 significant or incidental pathological conditions of seven organ systems and 19 parasitic, viral, bacterial, or fungal agents. Several voles captured in the wild had fungal osteomyelitis of the tail that disseminated systemically in a vole brought from the wild to the colony and may have been caused by a Penicillium sp. Three voles reintroduced from the colony to the wild experienced inanition and subsequent severe hepatic and moderate renal tubular lipidosis. The most common significant pathological conditions in colony-reared voles were chronic interstitial nephritis with proteinosis; cardiomyopathy; trichobezoars that, in intestines or cecocolic junctions, sometimes induced local rupture or infarction with peritonitis; multifocal gastrointestinal ulceration and colibacillosis; acute renal tubular necrosis or nephritis; sepsis; hepatic and renal lipidosis; molar apical elongation sometimes progressing to invasion of the calvarium; and mammary tumors. Uncommon diagnoses included intervertebral disc disease; microvascular dysplasia; and multifocal bacterial abscessation. Common or clinically important infectious agents included Demodex sp. mites in hair follicles, Demodex sp. in esophageal mucosa, and an outbreak of tropical rat mites thought to have been introduced via the straw bedding; gastrointestinal Helicobacter sp.; attaching and effacing Escherichia coli ; and Citrobacter braakii , a possible zoonotic bacterium. This survey of species-specific diseases and pathogens was possible because the established health surveillance program that is part of the species recovery plan allowed for monitoring of voles throughout the duration of their natural life spans in captivity.

The liver fluke Fasciola hepatica (Trematoda: Fasciolidae) causes fascioliasis, which affects mostly domestic ruminants and humans worldwide. This parasite has an Old World origin and was introduced into the New World by European colonizers. Capybara ( Hydrochoerus hydrochaeris) is the largest living rodent species, with adults weighing over 60 kg. We report a fascioliasis outbreak caused by F. hepatica that reduced a capybara group from 21 to 2 animals within a 9-mo period. Animal infection and associated lesions were confirmed by postmortem examinations that revealed extensive liver damage associated with the presence of large number of adult and immature forms of F. hepatica. Both macroscopic and microscopic alterations in the liver were compatible with acute fascioliasis, which is characterized by a large parasite burden in the liver. Taxonomic identification of flukes collected from capybara livers were confirmed by molecular methods, which generated a mitochondrial NADH dehydrogenase I (NDI) gene partial sequence that was 100% identical to a F. hepatica NDI sequence from the United Kingdom. This is the first report of deleterious effects caused by F. hepatica in capybaras, highlighting the potential harm caused by this exotic parasite in the capybara.

Background: Parasite evolution is hypothesized to select for levels of parasite virulence that maximise transmission success. When host population densities fluctuate, low levels of virulence with limited impact on the host are expected, as this should increase the likelihood of surviving periods of low host density. We examined the effects of Morogoro arenavirus on the survival and recapture probability of multimammate mice (Mastomys natalensis) using a seven-year capture-mark-recapture time series. Mastomys natalensis is the natural host of Morogoro virus and is known for its strong seasonal density fluctuations., Results: Antibody presence was negatively correlated with survival probability (effect size: 5-8% per month depending on season) but positively with recapture probability (effect size: 8%)., Conclusions: The small negative correlation between host survival probability and antibody presence suggests that either the virus has a negative effect on host condition, or that hosts with lower survival probability are more likely to obtain Morogoro virus infection, for example due to particular behavioural or immunological traits. The latter hypothesis is supported by the positive correlation between antibody status and recapture probability which suggests that risky behaviour might increase the probability of becoming infected.

Background: In the regular wildlife monitoring action carried out in the summer of the past few years at the Berlenga Island, wild rabbits (Oryctolagus cuniculus) have been repeatedly found dead. However, the origin of those deaths was never investigated. Our aim was to investigate the cause of death of 11 rabbits collected between April and May 2016., Results: While screening samples from rabbit carcasses for the major viral rabbit pathogens, five tested positive to RHDV2 but all were negative for RHDV and myxoma virus (MYXV). For six RHDV2-negative specimens, emaciation and parasitism were considered the most probable cause of death. Lesions identified in the RHDV2-positive rabbits included non-suppurative diffuse hepatic necrosis and pulmonary lesions varying from congestion and oedema of the lungs to interstitial pneumonia. Sequencing analysis of the vp60 gene obtained from two specimens showed identical vp60 sequences. Comparison with other known RHDV2 strains from public databases through BLAST analysis revealed a closer similarity with strains from Alentejo collected during 2013. Maximum Likelihood and Bayesian phylogenetic analysis showed that the 2016 strains from the archipelago have a higher resemblance with a group of strains mostly collected in the South of Portugal between 2013 and 2014., Conclusion: The results suggest that RHDV2 may have been introduced on the Berlenga Island a few years ago, having evolved separately from mainland strains due to insularity.

Marburg virus (MARV) causes severe, often fatal, disease in humans and transient illness in rodents. Sequential passaging of MARV in guinea pigs resulted in selection of a lethal virus containing 4 aa changes. A D184N mutation in VP40 (VP40D184N), which leads to a species-specific gain of viral fitness, and three mutations in the active site of viral RNA-dependent RNA polymerase L, which were investigated in the present study for functional significance in human and guinea pig cells. The transcription/replication activity of L mutants was strongly enhanced by a substitution at position 741 (S741C), and inhibited by other substitutions (D758A and A759D) in both species. The polymerase activity of L carrying the S741C substitution was eightfold higher in guinea pig cells than in human cells upon co-expression with VP40D184N, suggesting that the additive effect of the two mutations provides MARV a replicative advantage in the new host.

Wild rodents such as Peromyscus spp. are intermediate hosts for the zoonotic ascarid Baylisascaris procyonis (raccoon roundworm), and previous studies indicate Peromyscus leucopus (white-footed mouse) likely serves an important role in parasite ecology. Natural infections have been sporadically identified in a few Peromyscus spp., but no data are available on differences in susceptibility among the many other species. We compared survival and infection dynamics of B. procyonis in 4 species ( P. leucopus , Peromyscus maniculatus [deer mouse], Peromyscus californicus [California mouse], Peromyscus polionotus [Oldfield mouse]) from regions of varying habitat types as well as B. procyonis prevalence in raccoons. Six captive-bred mice of each species were inoculated per os with 1 of 3 biologically-relevant doses of embryonated B. procyonis eggs (∼10, ∼50, or ∼500). Animals were monitored twice daily for clinical signs and behavioral abnormalities and were euthanized at the onset of neurological signs or extensive (≥20%) weight loss, or at 45 days post-infection if no disease developed. Larvae were counted in the brain via microscopic examination and in skeletal muscle and visceral organs via artificial digestion. In the high-dose group, all but 1 mouse developed severe neurologic disease and were euthanized. In the medium-dose group, survival was variable and ranged from 33-85% across species. Little to no disease was observed in the low-dose group, although 1 P. maniculatus developed disease and was euthanized. Survival analysis reveals P. leucopus had a longer time until clinical disease onset versus the other species, which did not differ significantly from each other. Interestingly, larval recovery relative to dose was nearly identical across species and doses; however, larvae were differentially distributed in skeletal muscle, visceral organs, and brain among species. These data indicate that P. leucopus may be more resilient toward severe baylisascariasis compared to the other species and that even closely-related rodents may experience differential mortality. This variation in tolerance may have ecological implications for the different species as B. procyonis intermediate hosts, although more work is needed to put these experimental findings into context.

In research studies using rats or mice, the cause of death is often not evaluated or reported. An analysis of the causes of death is particularly valuable for aging and carcinogenesis studies. Comparing causes of death among the study groups is often an important adjunct to the biochemical, molecular, clinical, and histopathologic findings. The methods for evaluating causes of death, contributing causes of death, and comorbidities have been suggested in several publications. Surprisingly, in important mouse aging studies, causes of death are often not reported. Cause-of-death assignment in preclinical rodent model aging research suffers from a lack of a standardized approach and an understanding of the value that it can add to longevity and interventional studies. While assigning single cause of death may facilitate data analysis, defining and publishing data on contributing causes (comorbidities) provides more information on associated underlying chronic conditions and health span in mouse models. This article reviews factors that affect determination of cause of death and the methods for evaluating causes of death and comorbidities. The proposed systematic pathology analysis includes assigning cause of death and comorbidities to define total disease burden. The combination of pathology with in vivo data will fully characterize the effect of tested interventions on multiple chronic diseases and health span of aging mice with improved translation to human aging and age-associated lesions., (© The Author(s) 2015.)

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.

Prairie dogs (Cynomys spp.) suffer high rates of mortality from plague. An oral sylvatic plague vaccine using the raccoon poxvirus vector (designated RCN-F1/V307) has been developed for prairie dogs. This vaccine is incorporated into palatable bait along with rhodamine B as a biomarker. We conducted trials in August and September 2012 to demonstrate uptake and apparent safety of the RCN-F1/V307 vaccine in two prairie dog species under field conditions. Free-ranging prairie dogs and other associated small rodents readily consumed vaccine-laden baits during field trials with no apparent adverse effects; most sampled prairie dogs (90%) and associated small rodents (78%) had consumed baits. Visual counts of prairie dogs and their burrows revealed no evidence of prairie dog decline after vaccine exposure. No vaccine-related morbidity, mortality, or gross or microscopic lesions were observed. Poxviruses were not isolated from any animal sampled prior to bait distribution or on sites that received placebo baits. We isolated RCN-F1/V307 from 17 prairie dogs and two deer mice (Peromyscus maniculatus) captured on sites where vaccine-laden baits were distributed. Based on these findings, studies examining the utility and effectiveness of oral vaccination to prevent plague-induced mortality in prairie dogs and associated species are underway.

Rift Valley fever virus (RVFV) is a formidable pathogen that causes severe disease and abortion in a variety of livestock species and a range of disease in humans that includes hemorrhagic fever, fulminant hepatitis, encephalitis and blindness. The natural transmission cycle involves mosquito vectors, but exposure can also occur through contact with infected fluids and tissues. The lack of approved antiviral therapies and vaccines for human use underlies the importance of small animal models for proof-of-concept efficacy studies. Several mouse and rat models of RVFV infection have been well characterized and provide useful systems for the study of certain aspects of pathogenesis, as well as antiviral drug and vaccine development. However, certain host-directed therapeutics may not act on mouse or rat pathways. Here, we describe the natural history of disease in golden Syrian hamsters challenged subcutaneously with the pathogenic ZH501 strain of RVFV. Peracute disease resulted in rapid lethality within 2 to 3 days of RVFV challenge. High titer viremia and substantial viral loads were observed in most tissues examined; however, histopathology and immunostaining for RVFV antigen were largely restricted to the liver. Acute hepatocellular necrosis associated with a strong presence of viral antigen in the hepatocytes indicates that fulminant hepatitis is the likely cause of mortality. Further studies to assess the susceptibility and disease progression following respiratory route exposure are warranted. The use of the hamsters to model RVFV infection is suitable for early stage antiviral drug and vaccine development studies.

There is a significant genetic diversity of Toxoplasma gondii in Brazil. Two parasite isolates were recently obtained from chickens in Uberlândia, Minas Gerais state, Brazil, namely, TgChBrUD1 and TgChBrUD2. In this study, we investigated Calomys callosus susceptibility to these atypical T. gondii strains. Male and female animals were intraperitoneally infected with tachyzoites and monitored to evaluate body weight change, morbidity, and mortality. Immunohistochemical assay and qPCR were performed to determine the parasitism in liver, spleen, and brain. Our data showed that TgChBrUD2-infected males died earlier than TgChBrUD1-infected males and 100% of mortality was observed after 10 and 12 days of infection, respectively. Also, TgChBrUD1-infected females died earlier than TgChBrUD1-infected males and 100% of mortality was observed after 9 and 12 days of infection, respectively. Both strains were able to induce a decrease in body weight of males, but only the TgChBrUD1 strain induced an increase in body weight of females. TgChBrUD2-infected females had significantly higher parasite load in both liver and spleen in comparison to TgChBrUD1-infected females, but no significant difference was found between genders or strains when males were infected. There was higher parasitism in the liver than the brain from both males and females infected with either strain. In conclusion, C. callosus specimens are susceptible to both T. gondii atypical strains with differences between males and females in severity of infection. These findings open new prospects for understanding different aspects of T. gondii infection, including reinfection and vertical transmission with these atypical strains when utilizing this experimental model.

Background: The red squirrel population in Great Britain has declined dramatically in recent decades, principally due to squirrelpox. Concern exists that red squirrels may become extinct nationally and, as there has been limited research in to diseases other than squirrelpox, this study aimed to identify additional causes of mortality., Results: Post-mortem examinations on 163 red squirrels found dead on Isle of Wight (IoW) England, in Scotland and at other locations in Great Britain showed that 41.7% (n = 68) were killed by road traffic and 9.2% (n = 15) by predators, principally domestic cats and dogs. The overall male/female ratio was 1.08/1. Fleas were recorded on 34.9% of IoW squirrels and on 43.8% of Scottish squirrels but sucking lice and ixodid ticks were only seen on Scottish squirrels. Bacterial infections were significant, particularly in association with respiratory disease (n = 16); two squirrels died of Bordetella bronchiseptica bronchopneumonia. Cases of fatal exudative dermatitis (n = 5) associated with a lukM-positive clone of Staphylococcus aureus occurred only on the IoW. Toxoplasmosis (n = 12) was also confined to IoW where it was responsible for almost one tenth (9.5%) of all deaths. Hepatozoonosis was common, especially in IoW squirrels, but was not considered a primary cause of mortality. Hepatic capillariasis affected four IoW squirrels and one from Scotland. Fungal infections included oral candidiasis, adiaspiromycosis and pulmonary phaeohyphomycosis. Neoplastic conditions diagnosed were: pulmonary carcinoma, gastric spindle cell tumour, renal papillary adenoma and trichoepithelioma. Epidermal hyperplasia of unknown aetiology was seen in squirrels showing crusty lesions of the ear pinnae on IoW (n = 3) and Brownsea Island (n = 1), associated in two cases with cutaneous wart-like growths. Miscellaneous diagnoses included chylothorax, electrocution, intussusception, suspected cholecalciferol rodenticide poisoning and foetal death and mummification. No cases of squirrelpox were diagnosed., Conclusions: Red squirrels in Britain suffer premature or unnatural mortality due to a number of conditions in addition to squirrelpox, many of which result, directly or indirectly, from human activities: road traffic trauma, pet predation, toxoplasmosis, trap injuries, rodenticide poisoning and electrocution accounted for 61% of all recorded mortality in this study. Red squirrels are also affected by several diseases of unknown aetiology which merit further research.

A detailed epidemiologic survey of spontaneous diseases of mice used in biomedical research has not been performed in more than 4 decades. The current study examined all mouse disease reports for a subset of the University of Pennsylvania vivaria from October 2010 through September 2011. Mortality logs were examined over the same period of time. After eliminating protocol-related cases, the incidence rates for more than 30 diseases were calculated in terms of number of cases per 1000 cages per month. The average daily census for the facilities analyzed exceeded 29,000 cages and included more than 180 research groups. No single research group accounted for more than 4% of the total number of cases reported, indicating that this study did not simply quantify the spontaneous disease incidence in a limited number of research groups. Spontaneous mortality with unknown cause in adult and neonatal mice without prior reported illness was the most commonly reported issue, followed by dermatitis, ocular disease, and nonspecific clinical signs including lethargy, poor hair coat, and muscle wasting. These results indicate that improving the ability to identify sick mice is important in refining the care and use of mice in biomedical research. The information provided in the current study can help to provide a baseline for comparison, guide the field in directing mouse welfare research toward areas of need, and identify optimal methods of care for mice in biomedical research.

It has been shown previously that fluoroquinolone resistance (defined by resistance to at least 2 mg/liter ofloxacin) has a different impact on moxifloxacin monotherapy depending on the mutation in the sole fluoroquinolone target in Mycobacterium tuberculosis, i.e., DNA gyrase. Since tuberculosis treatment relies on multidrug therapy, we wished to determine the impact of fluoroquinolone resistance on the bactericidal and sterilizing activity of a second-line antituberculous regimen containing moxifloxacin. A total of 280 mice were inoculated with the wild-type Mycobacterium tuberculosis H37Rv strain or one of 3 isogenic fluoroquinolone-resistant mutant strains with increasing moxifloxacin resistance (the GyrB D500N, GyrA A90V, and GyrA D94G strains) and then treated for 6 months with a second-line regimen containing moxifloxacin, pyrazinamide, and ethionamide supplemented with amikacin during the first 2 months. Mice were sacrificed during treatment for measurement of bactericidal activity and 3 months after treatment completion for measurement of relapse rates (sterilizing activity). The CFU counts decreased faster in mice inoculated with the wild type than in mice inoculated with the mutant strains. The relapse rate after treatment completion was different among mice inoculated with mutant strains in relation to the drug resistance level: wild type, 0%; GyrB D500N strain, 33%; GyrA A90V strain, 50%; and GyrA D94G strain, 86%. The relapse rate observed with the GyrB D500N strain was the only one not statistically different from that observed with the wild-type strain. We demonstrated that the impact on sterilizing activity of the most active second-line drug regimen containing moxifloxacin depends on the MIC of moxifloxacin. We suggest that the precise level of moxifloxacin resistance be determined for all strains resistant to 2 mg/liter ofloxacin.

Plague, caused by the bacterium Yersinia pestis, triggers die-offs in colonies of black-tailed prairie dogs (Cynomys ludovicianus), but the time-frame of plague activity is not well understood. We document plague activity in fleas from prairie dogs and their burrows on three prairie dog colonies that suffered die-offs. We demonstrate that Y. pestis transmission occurs over periods from several months to over a year in prairie dog populations before observed die-offs.

Naked mole rats (NMRs; Heterocephalus glaber) are highly adapted, subterranean, eusocial rodents from semiarid regions of the eastern horn of Africa and the longest-living rodent known with a maximum life span of up to 30 years. They are a unique model for aging research due to their physiology, extreme longevity, and, when compared to mice and rats, resistance to cancer. Published surveys of disease in NMRs are sparse. Captive colonies in zoological collections provide an opportunity to monitor spontaneous disease over time in a seminatural environment. This retrospective study describes common lesions of a zoo population over a 15-year period during which 138 adult NMRs were submitted for gross and histologic evaluation. Of these, 61 (44.2%) were male, 77 (55.8%) female, 45 (32.6%) died, and 93 (67.4%) were euthanized. The most frequent cause of death or reason for euthanasia was conspecific trauma (bite wounds) and secondary complications. Some common histologic lesions and their prevalence were renal tubular mineralization (82.6%), hepatic hemosiderosis (64.5%), bite wounds (63.8%), chronic progressive nephropathy (52.9%), and calcinosis cutis (10.1%). In sum, 104 (75.4%) NMRs had more than one of the most prevalent histologic lesions. No malignant neoplasms were noted; however, there was a case of renal tubular adenomatous hyperplasia with nuclear atypia and compression that in rats is considered a preneoplastic lesion. This retrospective study confirms the NMR's relative resistance to cancer in spite of development of other degenerative diseases and highlights the utility of zoological databases for baseline pathological data on nontraditional animal models.

Proper protein folding in the endoplasmic reticulum (ER) is vital in all eukaryotes. When misfolded proteins accumulate in the ER lumen, the transmembrane kinase/endoribonuclease Ire1 initiates splicing of HAC1 mRNA to generate the bZIP transcription factor Hac1, which subsequently activates its target genes to increase the protein-folding capacity of the ER. This cellular machinery, called the unfolded protein response (UPR), is believed to be an evolutionarily conserved mechanism in eukaryotes. In this study, we comprehensively characterized mutant phenotypes of IRE1 and other related genes in the human fungal pathogen Candida glabrata. Unexpectedly, Ire1 was required for the ER stress response independently of Hac1 in this fungus. C. glabrata Ire1 did not cleave mRNAs encoding Hac1 and other bZIP transcription factors identified in the C. glabrata genome. Microarray analysis revealed that the transcriptional response to ER stress is not mediated by Ire1, but instead is dependent largely on calcineurin signaling and partially on the Slt2 MAPK pathway. The loss of Ire1 alone did not confer increased antifungal susceptibility in C. glabrata contrary to UPR-defective mutants in other fungi. Taken together, our results suggest that the canonical Ire1-Hac1 UPR is not conserved in C. glabrata. It is known in metazoans that active Ire1 nonspecifically cleaves and degrades a subset of ER-localized mRNAs to reduce the ER load. Intriguingly, this cellular response could occur in an Ire1 nuclease-dependent fashion in C. glabrata. We also uncovered the attenuated virulence of the C. glabrata Δire1 mutant in a mouse model of disseminated candidiasis. This study has unveiled the unique evolution of ER stress response mechanisms in C. glabrata.

The authors provide an update to their 2006 report on the successful large-scale captive breeding of the 13-lined ground squirrel (TLGS; Ictidomys tridecemlineatus) in their colony, now a single-source supplier of purpose-bred TLGSs for several research institutions. With funding from the National Institutes of Health, the authors have expanded their capacity for breeding pairs and built a dedicated hibernaculum to house torpid animals in the facility. The authors report new information on housing, diet, environmental enrichment, breeding, behavior and health. They also report the findings of several in-house studies on the health, behavior and well-being of their TLGSs.

In 2009 and 2010 16 Norwegian Eurasian beavers (Castor fiber) were reintroduced to Knapdale, Scotland as part of a 5-yr reintroduction trial (Scottish Beaver Trial). Despite numerous reintroduction programs throughout Europe there is no published information concerning recommended health surveillance during beaver reintroduction and only one publication describing causes of mortality. We describe the establishment of a health surveillance program based on International Union of Conservation of Nature (IUCN) and governmental guidelines, and report preliminary results based on the fecal and blood samples following the completion of the first stage of reintroduction. Animals underwent at least one general anesthetic to allow collection of fecal and blood samples and a thorough clinical examination. No bacterial enteric pathogens such as Salmonella spp., Campylobacter spp., or Yersinia pseudotuberculosis were isolated, nor were Giardia spp. or Cryptosporidium spp. However, numerous helminths including Travassosius rufus and Stichorchis subtriquetrus were detected. Five animals were positive for Leptospira antibodies. This included Leptospira saxkoebing, Leptospira canicola, Leptospira copenhageni, Leptospira icterohaemorrhagiae, Leptospira autumnalis, and Leptospira javanica. The highest loss of animals (20%) was during the statutory 6-mo rabies quarantine period. No common cause of death was determined. The rabies quarantine conditions were waived for four remaining animals, three of which were introduced to the wild successfully. The authors recommend the shortest possible quarantine period when introducing beavers, but allowing for the minimum recommended IUCN 35 days to allow for implementation of the initial stage of the health surveillance program, examination of animals, sample collection, and processing.

PCR was used to amplify adenoviral DNA, and transmission electron microscopy (TEM) to detect adenovirus particles in tissue and intestinal content samples from red squirrels (Sciurus vulgaris) associated with a reintroduction study on Anglesey (North Wales), from other populations on the island and from stock held at the Welsh Mountain Zoo, 38&emsp14;km to the east. Samples were collected during the routine surveillance postmortem examinations of all 60 red squirrels with carcases retrieved in a suitable condition between 2004 and 2010, including 29 captive and 31 free-living animals. Following significant clusters of mortality in captive red squirrels, adenovirus was identified retrospectively in faecal material from 12 of 13 (92 per cent) examined carcases from squirrels captive on Anglesey, and 14 of 16 (88 per cent) from the Welsh Mountain Zoo. Virus was identified in 13 of 31 (42 per cent) free-living wild animals, with evidence of both subclinical and clinically significant enteric adenoviral infections in wild squirrels. Without ancillary PCR and TEM testing, the extent of adenovirus infection in such populations would have been underestimated. Screening protocols that include examinations for adenovirus should, therefore, be part of the routine biosecurity measures protecting reintroduction or captive breeding programmes for red squirrels.

How pathogens affect their hosts is a key question in infectious disease ecology, and it can have important influences on the spread and persistence of the pathogen. Sin Nombre virus (SNV) is the etiological agent of hantavirus pulmonary syndrome (HPS) in humans. A better understanding of SNV in its reservoir host, the deer mouse, could lead to improved predictions of the circulation and persistence of the virus in the mouse reservoir, and could help identify the factors that lead to increased human risk of HPS. Using mark-recapture statistical modeling on longitudinal data collected over 15 years, we found a 13.4% decrease in the survival of male deer mice with antibodies to SNV compared to uninfected mice (both male and female). There was also an additive effect of breeding condition, with a 21.3% decrease in survival for infected mice in breeding condition compared to uninfected, non-breeding mice. The data identified that transmission was consistent with density-dependent transmission, implying that there may be a critical host density below which SNV cannot persist. The notion of a critical host density coupled with the previously overlooked disease-induced mortality reported here contribute to a better understanding of why SNV often goes extinct locally and only seems to persist at the metapopulation scale, and why human spillover is episodic and hard to predict.

Chagas disease is a zoonosis caused by the flagellated parasite Trypanosoma cruzi and transmitted by triatomine insects to several mammalian species acting as reservoir hosts. In the present study, we assess T. cruzi-prevalence, survivorship and T. cruzi-infection rate of the endemic rodent Octodon degus from a hyper-endemic area of Chagas disease in Chile. Parasite detection is performed by PCR assays on blood samples of individuals captured in austral summer of 2010, and on non-infected individuals recaptured in 2011 as well as on new captures. Results show a high infection level in this species (up to 70%). Infected O. degus have the same chance of surviving to the next reproductive season as uninfected individuals, irrespective of sex. We suggest that O. degus, an abundant long-lived rodent with high dispersal capability, could be considered an important native reservoir of T. cruzi in the wild transmission cycle of Chagas disease in Chile., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Many zoonotic diseases are caused by rodent-borne viruses. Major fluctuations in the transmission of these viruses have been related to large changes in reservoir host population numbers due to external factors. However, the impact of the pathogen itself on the demography of its reservoir host is often overlooked. We investigated the impact of Puumala virus (PUUV) on survival and reproductive maturation probability of its reservoir host, the bank vole (Myodes glareolus). Three years (2004-06) of data from nine independent sites in southern Belgium were collected and analyzed with a capture-mark-recapture (CMR) method that includes statistical correction for the variation in capture probability of voles. A multistate model based on four states of reproductive activity and PUUV immunoglobulin G (IgG) antibody status was used to estimate survival and probability of transition from one reproductive or infection state to another. Although survival estimates for reproductively active voles were similar between infected and noninfected individuals, PUUV infection in reproductively inactive voles decreased mean monthly survival by 14%. PUUV infection was associated with a threefold increase in the probability of reproductive maturation in bank voles. Moreover, the probability of PUUV IgG seroconversion was three times higher for reproductively active voles compared to reproductively inactive voles. Our model indicates that PUUV infection may alter bank vole population dynamics by affecting both survival and maturation in its host. Additional studies, using CMR methodology with shorter time intervals between trapping sessions and possibly a longer duration, are needed to confirm these findings.

Pandemic A (H1N1) 2009 influenza virus (pH1N1) infection in pregnant women can be severe. The mechanisms that affect infection outcome in this population are not well understood. To address this, pregnant and nonpregnant BALB/c mice were inoculated with the wild-type pH1N1 strain A/California/04/09. To determine whether innate immune responses are associated with severe infection, we measured the innate cells trafficking into the lungs of pregnant versus nonpregnant animals. Increased infiltration of pulmonary neutrophils and macrophages strongly correlated with an elevated mortality in pregnant mice. In agreement with this, the product of nitric oxide (nitrite) and several cytokines associated with recruitment and/or function of these cells were increased in the lungs of pregnant animals. Surprisingly, increased mortality in pregnant mice was not associated with higher virus load because equivalent virus titers and immunohistochemical staining were observed in the nasal cavities or lungs of all mice. To determine whether exacerbated inflammatory responses and elevated cellularity resulted in lung injury, epithelial regeneration was measured. The lungs of pregnant mice exhibited reduced epithelial regeneration, suggesting impaired lung repair. Despite these immunologic alterations, pregnant animals demonstrated equivalent percentages of pulmonary influenza virus-specific CD8(+) T lymphocytes, although they displayed elevated levels of T-regulator lymphocytes (Tregs) in the lung. Also, pregnant mice mounted equal antibody titers in response to virus or immunization with a monovalent inactivated pH1N1 A/California/07/09 vaccine. Therefore, immunopathology likely caused by elevated cellular recruitment is an implicated mechanism of severe pH1N1 infection in pregnant mice.

Murray Valley encephalitis virus (MVEV) is a mosquito-borne flavivirus endemic to Australia and Papua New Guinea. Most strains of MVEV cause potentially fatal cases of encephalitis in humans and horses, and have been shown to be highly neuroinvasive in weanling mice. In contrast, the naturally occurring subtype Alfuy virus (ALFV) has never been associated with human disease, nor is it neuroinvasive in weanling mice, even at high doses. To identify viral factors associated with ALFV attenuation, a chimeric infectious clone was constructed containing the structural genes premembrane (prM) and envelope (E) of ALFV swapped into the MVEV genome. The resulting virus (vMVEV/ALFVstr) was no longer neuroinvasive in mice, suggesting that motifs within prM-E of ALFV confer attenuation. To define these motifs further, mutants were constructed by targeting divergent sequences between the MVEV and ALFV E proteins that are known markers of virulence in other encephalitic flaviviruses. MVEV mutants containing a unique ALFV sequence in the flexible hinge region (residues 273-277) or lacking the conserved glycosylation site at position 154 were significantly less neuroinvasive in mice than wild-type MVEV, as determined by delayed time to death or increased LD(50). Conversely, when the corresponding MVEV sequences were inserted into the vMVEV/ALFVstr chimera, the mutant containing the MVEV hinge sequence was more neuroinvasive than the parental chimera, though not to the same level as wild-type MVEV. These results identify the hinge region and E protein glycosylation as motifs that contribute to the attenuation of ALFV.

H5N1 highly pathogenic avian influenza viruses (HPAIV) of clade 2.2 spread from Southeast Asia to Europe. Intriguingly, in contrast to all common avian strains specifying glutamic acid at position 627 of the PB2 protein (PB2-627E), they carry a lysine at this position (PB2-627K), which is normally found only in human strains. To analyze the impact of this mutation on the host range of HPAIV H5N1, we altered PB2-627K to PB2-627E in the European isolate A/Swan/Germany/R65/2006 (R65). In contrast to the parental R65, multicycle growth and polymerase activity of the resulting mutant R65-PB2(K627E) were considerably impaired in mammalian but not in avian cells. Correspondingly, the 50% lethal dose (LD₅₀) in mice was increased by three orders of magnitude, whereas virulence in chicken remained unchanged, resulting in 100% lethality, as was found for the parental R65. Strikingly, R65-PB2(K627E) reverted to PB2-627K after only one passage in mice but did not revert in chickens. To investigate whether additional R65 genes influence reversion, we passaged R65-PB2(K627E) reassortants containing genes from A/Hong Kong/156/97 (H5N1) (carrying PB2-627E), in avian and mammalian cells. Reversion to PB2-627K in mammalian cells required the presence of the R65 nucleoprotein (NP). This finding corresponds to results of others that during replication of avian strains in mammalian cells, PB2-627K restores an impaired PB2-NP association. Since this mutation is apparently not detrimental for virus prevalence in birds, it has not been eliminated. However, the prompt reversion to PB2-627K in MDCK cells and mice suggests that the clade 2.2 H5N1 HPAIV may have had a history of intermediate mammalian hosts.

West Nile virus (WNV) is a member of the family Flaviviridae and is a neurotropic pathogen responsible for severe human disease. Flavivirus-infected cells release virus particles that contain variable numbers of precursor membrane (prM) protein molecules at the viral surface. Consequently, antibodies are produced against the prM protein. These antibodies have been shown to activate the infectious potential of fully immature flavivirus particles in vitro. Here, we provide in vivo proof that prM antibodies render immature WNV infectious. Infection with antibody-opsonized immature WNV particles caused disease and death of mice, and infectious WNV was found in the brains and sera.

In an effort to evaluate novel derivatives from artemisinin, possessing potential antimalarial activity, a new derivative artecyclopentyl mether (CPM-1) was derivatized and evaluated for its dose-dependent efficacy in Plasmodium yoelii nigeriensis infected mice. The survivability of mice at 7.5 mg/kg was >28 days with negligible parasitaemia and recovered anemia (66.16-72.62%). Artecyclopentyl mether was also found to modulate the pro- and anti-inflammatory cytokines (IFN-γ, 39.64-56.92%; TNF, 49.10-74.31%; IL-4, 11.53-43.22%; IL-10, 37.60-53.52%) favourably besides optimizing the oxidative stress to the infected subjects as evident by the nitric oxide (88.76-95.43%), lipid peroxidation (59.30-76.05%) and glycaemic data (62.70-76.66%). The results indicate the potentiality of the new derivative as an antimalarial against asexual stages of the parasite.

Capillaria hepatica (syn. for Calodium hepaticum) is a zoonotic nematode parasitizing in the livers of rodents as main hosts and in numerous other mammals including humans. It is the causative agent of the rare conditions of hepatic capillariosis and spurious C. hepatica infections in humans. In this review, 163 reported cases of infestations with this parasite (72 reports of hepatic capillariosis, 13 serologically confirmed infestations and 78 observations of spurious infections) are summarized with an overview on the distribution, symptoms, pathology, diagnosis, serology and therapy of this rare human pathogen.

Streptococcus suis serotype 2 (S. suis 2) is an important porcine and human pathogen. Some proteins secreted by S. suis 2 are thought to play important roles in the pathogenesis of this organism and in its induced immune response. SsPepO has been previously identified as a secretary immunogenic protein using immunoproteomic techniques. In this study, we confirmed that the sequence of this protein is highly conserved in S. suis 2 and compared it with its homologues in other pathogens. To test the protective efficacy of SsPepO in animal models, the recombinant SsPepO protein was used to immunize mice and pigs. The results demonstrated that it could elicit a strong humoral antibody response and confer significant protection against challenge with a lethal dose of S. suis 2 in mice and pig models. In addition, the antisera against rSsPepO could efficiently inhibit bacterial growth in a whole blood assay and conferred significant protection against S. suis 2 infection in passive immunization experiments. Our findings suggest that SsPepO plays an important role in the pathogenesis of S. suis 2 and would be a promising subunit vaccine candidate., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In the past years, a significant rise in the proportion of childhood complicated pneumonia cases related to pneumococcal serotypes 1 and 3 has been observed. PhtD is a vaccine candidate protein antigen. By using a pneumococcal lethal intranasal challenge mouse model, a significant additive effect on protection was observed with the combination of vaccination-induced anti-PhtD and injected anti-polysaccharide antibodies specific for serotypes 1 and 3., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Heat shock proteins serve as important antigens in defense against infectious diseases. Members of HSP70 family, particularly microbial HSP70s have acquired special significance in immunity. In the present study, we evaluated the immunogenicity and protective efficacy of HSP70 of Salmonella enterica serovar Typhi against lethal infection by Salmonella in mice with or without adjuvants. The HSP70 gene was cloned and expressed in Escherichia coli BL21 and purified by affinity chromatography. Immunization of mice with HSP70 either alone or in combination with complete Freund's adjuvant (CFA) resulted in a significant increase in antibody titers as compared to control. Antibody isotyping revealed that HSP70 immunization induces both IgG1 and IgG2a antibodies to a significant extent but a higher IgG1/IgG2a ratio indicates a predominant Th2 response. There was a significant increase in lymphocyte proliferation, and levels of both Th2 and Th1 cytokines in cells isolated from immunized mice as compared to control. Immunization of mice with recombinant HSP70 either alone or in combination with CFA conferred 70-90% protection against lethal infections by Salmonella Typhi Ty2 or Salmonella Typhimurium. However, passive immunization with anti-HSP70 sera induced only partial protection in the immunized mice., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Whole-body bioimaging was employed to study the effects of passive immunotherapies on lethality and viral dissemination in BALB/c mice challenged with recombinant vaccinia viruses expressing luciferase. WRvFire and IHD-J-Luc vaccinia viruses induced lethality with similar times to death following intranasal infection, but WRvFire replicated at higher levels than IHD-J-Luc in the upper and lower respiratory tracts. Three types of therapies were tested: licensed human anti-vaccinia virus immunoglobulin intravenous (VIGIV); recombinant anti-vaccinia virus immunoglobulin (rVIG; Symphogen, Denmark), an investigational product containing a mixture of 26 human monoclonal antibodies (HuMAbs) against mature virion (MV) and enveloped virion (EV); and HuMAb compositions targeting subsets of MV or EV proteins. Bioluminescence recorded daily showed that pretreatment with VIGIV (30 mg) or with rVIG (100 μg) on day -2 protected mice from death but did not prevent viral replication at the site of inoculation and dissemination to internal organs. Compositions containing HuMAbs against MV or EV proteins were protective in both infection models at 100 μg per animal, but at 30 μg, only anti-EV antibodies conferred protection. Importantly, the t statistic of the mean total fluxes revealed that viral loads in surviving mice were significantly reduced in at least 3 sites for 3 consecutive days (days 3 to 5) postchallenge, while significant reduction for 1 or 2 days in any individual site did not confer protection. Our data suggest that reduction of viral replication at multiple sites, including respiratory tract, spleen, and liver, as monitored by whole-body bioluminescence can be used to predict the effectiveness of passive immunotherapies in mouse models.

Ebola virus is the etiologic agent of a lethal hemorrhagic fever in humans and nonhuman primates with mortality rates of up to 90%. Previous studies with Zaire Ebola virus (ZEBOV), mouse-adapted virus (MA-ZEBOV), and mutant viruses (ZEBOV-NP(ma), ZEBOV-VP24(ma), and ZEBOV-NP/VP24(ma)) allowed us to identify the mutations in viral protein 24 (VP24) and nucleoprotein (NP) responsible for acquisition of high virulence in mice. To elucidate specific molecular signatures associated with lethality, we compared global gene expression profiles in spleen samples from mice infected with these viruses and performed an extensive functional analysis. Our analysis showed that the lethal viruses (MA-ZEBOV and ZEBOV-NP/VP24(ma)) elicited a strong expression of genes 72 h after infection. In addition, we found that although the host transcriptional response to ZEBOV-VP24(ma) was nearly the same as that to ZEBOV-NP/VP24(ma), the contribution of a mutation in the NP gene was required for a lethal phenotype. Further analysis indicated that one of the most relevant biological functions differentially regulated by the lethal viruses was the inflammatory response, as was the induction of specific metalloproteinases, which were present in our newly identify functional network that was associated with Ebola virus lethality. Our results suggest that this dysregulated proinflammatory response increased the severity of disease. Consequently, the newly discovered molecular signature could be used as the starting point for the development of new drugs and therapeutics. To our knowledge, this is the first study that clearly defines unique molecular signatures associated with Ebola virus lethality.

The HA protein of the 2009 pandemic H1N1 viruses (H1N1pdm) is antigenically closely related to the HA of classical North American swine H1N1 influenza viruses (cH1N1). Since 1998, through mutation and reassortment of HA genes from human H3N2 and H1N1 influenza viruses, swine influenza strains are undergoing substantial antigenic drift and shift. In this report we describe the development of a novel monoclonal antibody (S-OIV-3B2) that shows high hemagglutination inhibition (HI) and neutralization titers not only against H1N1pdm, but also against representatives of the α, β, and γ clusters of swine-lineage H1 influenza viruses. Mice that received a single intranasal dose of S-OIV-3B2 were protected against lethal challenge with either H1N1pdm or cH1N1 virus. These studies highlight the potential use of S-OIV-3B2 as effective intranasal prophylactic or therapeutic antiviral treatment for swine-lineage H1 influenza virus infections., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Abdominal angiostrongyliasis (AA) is caused by the nematode Angiostrongylus costaricensis. Parasite-associated thrombosis of mesenteric vessels may lead to intestinal infarction, which might be prevented with anti-thrombotic agents. This study assessed the effect of enoxaparin on survival and pathological findings in Swiss mice with AA. In this experiment, 24 mice were infected with A. costaricensis (10 L3 per animal) followed by treatment with subcutaneous enoxaparin (40 mg/kg/day) or water (sham), starting from 15 days post-infection (dpi) and continued until animal death. Animals were monitored until death or sacrifice at the 50th dpi. Ten mice (42%) were dead after 36 ± 8 dpi. Of these, five (50%) were treated with enoxaparin. Animals treated with enoxaparin and sham did not differ in terms of weight loss (median, 1.3 vs. 4.2 g; P = 0.303) and macroscopical findings. Microscopically, no difference was found in regard to vascular granuloma (median grade, 2 vs. 3; P = 0.293) and presence of either vasculitis (75% vs. 100%; P = 0.217), mesenteric thrombosis (33% vs. 50%; P = 0.680), or bowel necrosis (25% vs. 50%; P = 0.400). Mice dead before the 50th dpi showed more pneumonia (90% vs. 21%; P = 0.002), bowel infarction (40% vs. 0%; P = 0.02), and purulent peritonitis (60% vs. 7%; P = 0.008) compared to survivors. Prophylactic enoxaparin in mice did not prevent tissue damage and mortality related with AA. The lower prevalence of mesenteric thrombosis and bowel infarction regardless of treatment were notorious. Frequent septic complications suggest the need of studies addressing the effect of antibiotics in AA.

During the last 2 decades there have been numerous reports of the emergence of mefloquine resistance in Southeast Asia and nearly 50% resistance is reported in Thailand. A World Health Organization report (2001) considers mefloquine as an important component of ACT (artesunate+mefloquine) which is the first line of treatment for the control of uncomplicated/multi-drug resistant (MDR) Plasmodium falciparum malaria. In view of the emergence of resistance towards this drug, it is proposed to develop new drug combinations to prolong the protective life of this drug. Prior studies have suggested that mefloquine resistance can be overcome by a variety of agents such as ketoconazole, cyproheptadine, penfluridol, Icajine and NP30. The present investigation reports that clarithromycin (CLTR), a new macrolide, being a potent inhibitor of Cyt. P450 3A4, can exert significant resistance reversal action against mefloquine resistance of plasmodia. Experiments were carried out to find out the curative dose of CLTR against multi-drug resistant P. yoelii nigeriensis. Mefloquine (MFQ) and clarithromycin (CLTR) combinations have been used for the treatment of this MDR parasite. Different dose combinations of these two drugs were given to the infected mice on day 0 (prophylactic) and day 1 with established infection (therapeutic) to see the combined effect of these combinations against the MDR malaria infection. With a dose of 32 mg/kg MFQ and 225 mg/kg CLTR, 100% cure was observed, while in single drug groups, treated with MFQ or CLTR, the cure was zero and 40% respectively. Therapeutically, MFQ and CLTR combinations 32+300 mg/kg doses cleared the established parasitaemia on day 10. Single treatment with MFQ or CLTR showed considerable suppression of parasitaemia on day 14 but neither was curative. Follow-up of therapeutically treated mice showed enhanced anti-malarial action as reflected by their 100% clearance of parasitaemia. The present study reveals that CLTR is a useful antibiotic to be used as companion drug with mefloquine in order to overcome mefloquine resistance in plasmodia.

We have characterized the csoR-copA-copZ copper resistance operon of the important human intracellular pathogen Listeria monocytogenes. Transcription of the operon is specifically induced by copper, and mutants lacking the P₁-type ATPase CopA have reduced copper tolerance and over-accumulate copper relative to wild type. The copper-responsive repressor CsoR autoregulates transcription by binding to a single 32 bp site spanning the -10 and -35 elements of the promoter. Copper co-ordination by CsoR derepresses transcription of the operon and alters CsoR:DNA complex assembly as determined by DNase I footprinting and electrophoretic mobility shift assays, with some DNA-binding capacity being retained in the presence of 2 mole equivalents of copper. Analysis of the CsoR copper sensory site demonstrated that substitution of Cys⁴² with Ala generated a CsoR variant that was unresponsive to copper. Importantly, in the absence of CopZ, copper responsiveness of csoR-copA-copZ expression is substantially increased, implying that CopZ reduces the access of CsoR to copper. Furthermore, CopZ is shown to confer copper resistance in mutants lacking copper-inducible csoR-copA-copZ expression, thus providing protection from the deleterious effects of copper within the cytoplasm., (© 2011 Blackwell Publishing Ltd.)

The immunological correlates for recovery from primary Japanese encephalitis virus (JEV) infection in humans and experimental animals remain poorly defined. To investigate the relative importance of the adaptive immune responses, we have established a mouse model for Japanese encephalitis in which a low-dose virus inoculum was administered into the footpads of adult C57BL/6 mice. In this model, ~60% of the mice developed a fatal encephalitis and a virus burden in the central nervous system (CNS). Using mice lacking B cells (μMT(-/-) mice) and immune B cell transfer to wild-type mice, we show a critically important role for humoral immunity in preventing virus spread to the CNS. T cell help played an essential part in the maintenance of an effective antibody response necessary to combat the infection, since mice lacking major histocompatibility complex class II showed truncated IgM and blunted IgG responses and uniformly high lethality. JEV infection resulted in extensive CD8(+) T cell activation, judged by upregulation of surface markers CD69 and CD25 and cytokine production after stimulation with a JEV NS4B protein-derived H-2D(b)-binding peptide and trafficking of virus-immune CD8(+) T cells into the CNS. However, no significant effect of CD8(+) T cells on the survival phenotype was found, which was corroborated in knockout mice lacking key effector molecules (Fas receptor, perforin, or granzymes) of cytolytic pathways triggered by T lymphocytes. Accordingly, CD8(+) T cells are mostly dispensable for recovery from infection with JEV. This finding highlights the conflicting role that CD8(+) T cells play in the pathogenesis of JEV and closely related encephalitic flaviviruses such as West Nile virus.

Background: Salmonella enterica serovar Typhimurium is an intracellular bacterial pathogen which can colonize a variety of hosts, including human, causing syndromes that vary from gastroenteritis and diarrhea to systemic disease., Results: In this work we present structural information as well as insights into the in vivo function of YqiC, a 99-residue protein of S. Typhimurium, which belongs to the cluster of the orthologous group 2960 (COG2960). We found that YqiC shares biophysical and biochemical properties with Brucella abortus BMFP, the only previously characterized member of this group, such as a high alpha helix content, a coiled-coil domain involved in trimerization and a membrane fusogenic activity in vitro. In addition, we demonstrated that YqiC localizes at cytoplasmic and membrane subcellular fractions, that a S. Typhimurium yqiC deficient strain had a severe attenuation in virulence in the murine model when inoculated both orally and intraperitoneally, and was impaired to replicate at physiological and high temperatures in vitro, although it was still able to invade and replicate inside epithelial and macrophages cell lines., Conclusion: This work firstly demonstrates the importance of a COG2960 member for pathogen-host interaction, and suggests a common function conserved among members of this group.

Citrobacter rodentium is a natural pathogen of mice that causes intestinal hyperplasia and colitis. Resistant strains such as C57BL/6J (B6) experience a self-limiting disease that peaks between one and two weeks post infection, followed by a clearing of the infection and complete recovery. However, the inbred mouse strains C3H/HeJ (C3), C3H/HeOuJ (C3Ou) and FVB/N (FVB) are highly susceptible to C. rodentium infection and develop more severe symptoms of disease leading to high rates of mortality during infection. We have recently demonstrated through a systematic genetics approach that a single locus on proximal chromosome 15 is responsible for the susceptibility of both C3 and C3Ou mice to C. rodentium infection. We have named the locus Citrobacter rodentium infection 1 (Cri1). Here we show that Cri1 also controls susceptibility to C. rodentium in FVB mice, using a targeted method of genotyping to stratify (B6 x FVB)F2 mice according to their genotype at Cri1. Mice that inherit two copies of the resistant B6 allele have 97% cumulative survival at day 30 post-infection, whereas those that inherit one or two copies of Cri1 from the FVB parent have significantly lower rates of survival (35% and 42%, respectively). These results provide evidence for a common genetic cause of fatal infectious colitis in C3, C3Ou and FVB mice following infection with Citrobacter rodentium.

An infectious disease caused by Squirrelpox virus has contributed to the decline of red squirrels, Sciurus vulgaris, in the British Isles. Because of the heightened disease surveillance activity in red squirrels, adenovirus infection with associated mortality has been detected. Adenoviral disease is described in other rodent species usually associated with stressors. Here we 1) describe the pathologic findings in red squirrels found dead with adenoviral infection and gastrointestinal disease, and 2) investigate the epizootiology of the disease through pathologic investigation, scanning surveillance, and virologic studies. Ten red squirrels involved in conservation studies were diagnosed with adenoviral infection by electron microscopy or PCR. All squirrels exhibited diarrhea and small intestinal inflammation or hemorrhage was evident in seven cases. Lesions indicative of splenic lymphocytolysis were observed in one squirrel and leukocytic hepatitis in another. No adenovirus was detected in grey squirrels, Sciurus carolinensis, inhabiting the same forest area, but previous serologic studies showed that grey squirrels cannot be discounted as a reservoir of the virus. Scanning surveillance showed that 12% of 493 red squirrels had diarrheal disease and two of 13 free-living red squirrels with diarrheal disease had adenovirus infection. Adenoviral disease in declining free-living wild red squirrel populations in the British Isles occurs at a detectable frequency and its impact on the conservation of this species deserves further attention.

Urtica dioica agglutinin (UDA) is a small plant monomeric lectin, 8.7 kDa in size, with an N-acetylglucosamine specificity that inhibits viruses from Nidovirales in vitro. In the current study, we first examined the efficacy of UDA on the replication of different SARS-CoV strains in Vero 76 cells. UDA inhibited virus replication in a dose-dependent manner and reduced virus yields of the Urbani strain by 90% at 1.1 ± 0.4 μg/ml in Vero 76 cells. Then, UDA was tested for efficacy in a lethal SARS-CoV-infected BALB/c mouse model. BALB/c mice were infected with two LD50 (575 PFU) of virus for 4 h before the mice were treated intraperitoneally with UDA at 20, 10, 5 or 0 mg/kg/day for 4 days. Treatment with UDA at 5 mg/kg significantly protected the mice against a lethal infection with mouse-adapted SARS-CoV (p < 0.001), but did not significantly reduce virus lung titers. All virus-infected mice receiving UDA treatments were also significantly protected against weight loss (p < 0.001). UDA also effectively reduced lung pathology scores. At day 6 after virus exposure, all groups of mice receiving UDA had much lower lung weights than did the placebo-treated mice. Thus, our data suggest that UDA treatment of SARS infection in mice leads to a substantial therapeutic effect that protects mice against death and weight loss. Furthermore, the mode of action of UDA in vitro was further investigated using live SARS-CoV Urbani strain virus and retroviral particles pseudotyped with SARS-CoV spike (S). UDA specifically inhibited the replication of live SARS-CoV or SARS-CoV pseudotyped virus when added just before, but not after, adsorption. These data suggested that UDA likely inhibits SARS-CoV infection by targeting early stages of the replication cycle, namely, adsorption or penetration. In addition, we demonstrated that UDA neutralizes the virus infectivity, presumably by binding to the SARS-CoV spike (S) glycoprotein. Finally, the target molecule for the inhibition of virus replication was partially characterized. When UDA was exposed to N-acetylglucosamine and then UDA was added to cells just prior to adsorption, UDA did not inhibit the virus infection. These data support the conclusion that UDA might bind to N-acetylglucosamine-like residues present on the glycosylated envelope glycoproteins, thereby preventing virus attachment to cells., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Marburgvirus (MARV) infections are generally lethal in humans and nonhuman primates but require in vivo lethal mouse variant selection by the serial transfer (passage) of the nonlethal virus into naïve mice to propagate a lethal infection. The passage of progenitor (wild-type) MARV or Ravn virus (RAVV) from infected scid BALB/c mouse liver homogenates into immunocompetent BALB/c mice results in the selection of lethal mouse viruses from within the quasispecies sufficient to establish lethality in immunocompetent mice. Genomic analysis in conjunction with the passage history of each mutation detailed the altered primary and secondary structures of the viral genomic RNA throughout the process. Key findings included the following: (i) a VP40:D184N mutation previously identified in the lethal guinea pig MARV genome was the first mutation to occur during the passage of both the MARV and RAVV variants; (ii) there was biased hypermutagenesis in the RAVV variant genome; (iii) there were two identical mutations in lethal mouse MARV and RAVV variants, VP40:Y19H in the PPPY motif and VP40:D184N in a loop structure between the two VP40 domains; (iv) the passage of wild-type MARV and RAVV in mice resulted in the selection of viral variants from among the quasispecies with different genotypes than those of the wild-type viruses; and (v) a lethal mouse RAVV variant had different tissue tropisms distinct from those of its wild-type virus. These studies provide insights into how marburgviruses manipulate the host for enzymes, metabolites, translation regulators, and effectors of the innate immune response to serve as potential viral countermeasures.

New and emerging influenza virus strains, such as the pandemic influenza A (H1N1) virus require constant vigilance for antiviral drug sensitivity and resistance. Efficacy of intramuscularly (IM) administered neuraminidase (NA) inhibitor, peramivir, was evaluated in mice infected with recently isolated pandemic A/California/04/2009 (H1N1, swine origin, mouse adapted) influenza virus. A single IM injection of peramivir (four dose groups), given 1h prior to inoculation, significantly reduced weight loss (p < 0.001) and mortality (p < 0.05) in mice infected with LD90 dose of pandemic A/California/04/2009 (H1N1) influenza virus compared to vehicle group. There was 20% survival in the vehicle-treated group, whereas in the peramivir-treated groups, survival increased in a dose-dependent manner with 60, 60, 90 and 100% survivors for the 1, 3, 10, and 30 mg/kg doses, respectively. Weight loss on day 4 in the vehicle-treated group was 3.4 gm, and in the peramivir-treated groups was 2.1, 1.5, 1.8 and 1.8 g for the 1, 3, 10 and 30 mg/kg dose groups, respectively. In the treatment model, peramivir given 24h after infection as a single IM injection at 50mg/kg dose, showed significant protection against lethality and weight loss. There was 13% survival in the vehicle-treated group while in the peramivir-treated group at 24, 48, and 72 h post infection, survival was 100, 40, and 50%, respectively. Survival in the oseltamivir groups (10 mg/kg/d twice a day, orally for 5 days) was 90, 30 and 20% at 24, 48 and 72 h, respectively. These data demonstrate efficacy of parenterally administered peramivir against the recently isolated pandemic influenza virus in murine infection models., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We determined the role of Yersinia pestis virulence markers in an animal model of pneumonic plague. Eleven strains of Y. pestis were characterized using PCR assays to detect the presence of known virulence genes both encoded by the three plasmids as well as chromosomal markers. The virulence of all Y. pestis strains was compared in a mouse model for pneumonic plague. The presence of all known virulence genes correlated completely with virulence in the Balb/c mouse model. Strains which lacked HmsF initially exhibited visible signs of disease whereas all other strains (except wild-type strains) did not exhibit any disease signs. Forty-eight hours post-infection, mice which had received HmsF(-) strains regained body mass and were able to control infection; those infected with strains possessing a full complement of virulence genes suffered from fatal disease. The bacterial loads observed in the lung and other tissues reflected the observed clinical signs as did the cytokine changes measured in these animals. We can conclude that all known virulence genes are required for the establishment of pneumonic plague in mammalian animal models, the role of HmsF being of particular importance in disease progression.

Background: Mannose-binding lectin (MBL) is an important component of innate immunity because it promotes bacterial clearance and neutralization of human influenza A viruses. Since a majority of humans have no neutralizing antibody against the pandemic (H1N1) 2009 influenza (pandemic 2009) virus, innate immunity may be crucial and MBL susceptibility may therefore influence viral pathogenesis., Results: We examined MBL susceptibility of influenza A viruses and observed that the pandemic 2009 virus was resistant to MBL, whereas all seasonal influenza A viruses tested were susceptible. The mortality of mice infected with a seasonal H1N1 influenza virus was evidently enhanced on transient blockage of MBL activity by simultaneous inoculation of mannan, whereas mannan inoculation had no effect on mice infected with a pandemic 2009 virus. This indicates that MBL protects mice against infection with the seasonal virus but not against that with the pandemic 2009 virus., Conclusions: These results indicate that the pandemic 2009 virus is not susceptible to MBL, an important component of innate immunity.