Evaluation of a Commercial Colorimetric Fecal Dipstick Assay for the Detection of Helicobacter hepaticus Infections in Laboratory Mice

Mice used in biomedical research typically are tested for the presence of Helicobacter spp., including Helicobacter hepaticus. Here we evaluated the ability of a commercially available colorimetric Helicobacter dipstick assay to detect H. hepaticus in experimentally and naturally infected mice, with use of a Helicobacter PCR assay as the ‘gold standard’ test. None of the fecal samples from experimentally infected A/JCr mice (n = 12) tested positive for Helicobacter by the colorimetric dipstick test. In naturally infected A/JCr and C57BL/6 mice, 11% (1 of 9) and 30% (3 of 10) of fecal samples, respectively, tested positive for Helicobacter by the colorimetric dipstick assay. In these 3 groups of H. hepaticus-infected mice, statistically fewer mice tested positive by the colorimetric dipstick test than by PCR. The colorimetric Helicobacter dipstick assay had an overall diagnostic sensitivity of 13%, diagnostic specificity of 94%, and analytical sensitivity of 10⁸ H. hepaticus cfu/mL. As currently formulated, the colorimetric dipstick assay had high specificity but lacked sensitivity for detecting H. hepaticus infections in 2 strains of mice commonly used in research, thereby limiting its utility as a diagnostic screening test for H. hepaticus infections in research mice.Recent surveys indicate that members of the genus Helicobacter are among the most often detected bacterial pathogens in the gastrointestinal tracts of laboratory mice.^4,9,13 At least 8 Helicobacter spp. naturally infect mice used in biomedical research.^8,16,17 Of these, Helicobacter hepaticus has been reported to be the most prevalent pathogenic species of Helicobacter detected in laboratory mouse colonies.^4,8,9 Consequences of infection with H. hepaticus range from subclinical disease to overt morbidity in susceptible mouse strains. In addition, hepatic and colonic neoplasia, hepatitis, and typhlocolitis have been associated with H. hepaticus infection in multiple strains of mice.^4,8,14,16,17 Two such strains, A/JCr and C57BL/6, differ in their response to H. hepaticus infections, with A/JCr mice susceptible and C57BL/6 mice resistant to hepatic and large intestinal pathology.¹⁵Identification of Helicobacter positive mice is an integral component of many health monitoring programs. Accurate detection of Helicobacter spp. is crucial to maintain Helicobacter-free mouse colonies and minimize the potential for these pathogens to confound research studies, especially those involving enterohepatic disease models.⁷ Diagnostic approaches to detect Helicobacter infections in mice include microbiologic culture of intestinal contents or feces, serologic assays for the detection of Helicobacter-specific antibodies in serum, histopathologic evaluation of liver or large bowel, and PCR testing of feces or tissues. Currently, evaluation of feces by PCR is the ‘gold standard’ diagnostic assay for detecting enteric Helicobacter infections in mice because these assays provide the highest sensitivity, specificity, and convenience for identifying Helicobacter infections.^1,2,12,17 Recently, an assay was made commercially available that is a one-step rapid screening test for qualitative detection of Helicobacter infections. The colorimetric assay uses an enzymatic reaction, and subsequent color change, to identify Helicobacter antigen in infected mouse feces. Although results can be obtained rapidly, data regarding the diagnostic performance of this assay are not available. The purpose of the present studies was to evaluate the diagnostic performance of the colorimetric assay by using a fecal PCR assay as the gold standard test for detecting Helicobacter infections in research mice. As such, the sensitivity and specificity of the colorimetric dipstick test was evaluated in A/JCr mice experimentally infected and C57BL/6 and A/JCr mice naturally infected with H. hepaticus, a highly prevalent, pathogenic enteric Helicobacter.     

Use of Neonatal Fostering To Remove Helicobacter spp. from Deer Mice (Peromyscus maniculatus)

Helicobacter species can be found in a wide variety of animals and remain common contaminants of laboratory rodents. Fostering of neonatal pups has been used to eliminate Helicobacter spp. from various laboratory rodents, including laboratory mice and gerbils. Deer mice (Peromyscus maniculatus) from a captive colony enzootic for at least one Helicobacter species were mated, and the pups produced were fostered on laboratory mice 24 h after birth. After 2 rounds of fostering, both foster dams and pups were free of Helicobacter spp. as determined by fecal PCR analysis. Removal of Helicobacter infection through neonatal fostering has not been described previously for Peromyscus maniculatus.Abbreviation: PGSC, Peromyscus Genetic Stock CenterThe deer mouse (Peromyscus maniculatus) is commonly found throughout North America and is so named because of its deer-like coloration and ability to jump.² Their ubiquity makes deer mice a useful mammal for assessing the effects of environmental stressors on organ systems and on reproductive capabilities through generational studies. Furthermore, deer mice are easy to maintain in captivity.² Among the permanent colonies established from wild-caught deer mice in North America, the oldest one is descended from animals originally collected in Washtenaw County, Michigan, in 1946 and 1947. About 1954, a substock was established at Ohio State University and, in 1962, the colony at the University of South Carolina was founded by using animals from Ohio State.⁹ The newest permanent deer mouse colony was established in 2013, using mice from the Peromyscus Genetic Stock Center (PGSC), at the Biologic Research Facility of the Chalk River Laboratories of Atomic Energy of Canada Limited in Chalk River, Ontario, Canada.In research, confounding factors must be kept to a minimum. It follows that pathogenic or potentially pathogenic microorganisms and parasites that are an undesired part of resident flora are eliminated if they can influence study results. In addition, pathogen-free animals do not pose a risk of infection to animals housed in the same facility for other studies. As a result, in modern laboratory facilities, most agents that cause widespread clinical disease in mice have long been excluded, and those that are left tend to cause either mild or inapparent infections.^3,15 Helicobacter is a potentially pathogenic bacterial genus that can confound research because of its association with gastric cancer in humans, chronic hepatitis in mice,^25,26 colitis in mice with or without rectal prolapse, and other conditions that may alter experimental results.⁴ It is common to discover members of the genus Helicobacter in both domestic and wild-derived rodent colonies, especially as new species of Helicobacter are characterized. Some species of Helicobacter have unknown pathogenic potential, but other helicobacters, especially in combination with specific mouse or rat genetic backgrounds, can have serious health consequences for animals.²⁷ Although a pharmaceutical therapy is available—and is often successful—rederivation is considered the most certain way to eliminate Helicobacter spp.¹⁰When Chalk River Laboratories decided to import P. maniculatus for research purposes, the presence of Helicobacter in the animals from PGSC meant that the mice did not meet the exclusion criteria already established for the Chalk River facility. Therefore mice from PGSC were shipped to Charles River Laboratories for an attempted rederivation.     

Eradication of Helicobacter spp. by Using Medicated Diet in Mice Deficient in Functional Natural Killer Cells and Complement Factor D

A commercial 4-drug diet has shown promise in eradicating Helicobacter spp. from rodents; however, its effectiveness in immunocompromised mice is unknown. This study evaluated the efficacy of this treatment in eradicating Helicobacter spp. from mice deficient in functional natural killer cells (Cd1^−/−) or complement factor D (Df ^−/−). Cd1^−/− mice naturally infected with H. hepaticus with or without H. rodentium were fed either control or medicated diet for 8 wk followed by 4 wk on control diet. Fecal samples were PCR-evaluated for Helicobacter spp. before mice began treatment and then every 2 wk thereafter for 12 wk. The same experimental design was repeated for eighteen 9- to 21-wk-old Df ^−/− mice naturally infected with H. bilis with or without H. rodentium. All Df ^−/− mice and 8- to 21-wk-old Cd1^−/− mice ceased shedding Helicobacter spp. after 2 wk of treatment and remained negative throughout the study. In contrast, the Cd1^−/− mice that were 24 wk or older shed Helicobacter spp. for the first 8 wk but tested negative at 10 and 12 wk. All treated animals had enlarged ceca and gained less weight than control untreated mice, and 6 of 7 treated Cd1^−/− male mice developed mild portal fibrosis. These findings show that within 2 wk of treatment, the 4-drug diet eradicated H. hepaticus and H. rodentium from young Cd1^−/− mice and H. bilis and H. rodentium from Df ^−/− mice, but eradication of established infections in Cd1^−/− mice required 8 wk of treatment.Helicobacter spp. infections are widespread within academic institutions, whereas most rodent vendors have successfully eliminated the bacteria from their stock.²⁵ Infections of Helicobacter hepaticus alone or in combination with other Helicobacter spp. are the most frequently diagnosed infections,³⁵ and species prevalence varies both by geographic location and by colony within individual institutions.^14,25,35The research implications of intercurrent infection with various Helicobacter spp. have recently been reviewed.⁶ For example, H. hepaticus was responsible for hepatitis and hepatic tumors in control mice on long-term carcinogenesis studies in A/JCr, SCID/NCr, and C3H/HeNCr⁴⁰ and B6C3F1 mice,¹⁵ and H. bilis infection caused hepatitis in outbred SW mice in a long-term oral supplementation study looking for organ-specific histologic lesions.¹¹ In addition, Helicobacter spp. have been implicated in the alteration of immunologic parameters, such as inhibition of oral tolerance.²¹ Mice with immune deficiencies often develop severe pathology: scid/Trp53^−/− mice developed typhlocolitis and proctitis when infected with H. bilis and H. typhlonius,⁴² and IL10^−/− mice developed reproductive problems when infected with H. typhlonius or H. rodentium.³¹ These and other examples demonstrate a need to eliminate Helicobacter spp. from infected mouse colonies, particularly those that are immunocompromised.To date the most successful methods of Helicobacter eradication have been labor-intensive. Methods that have proven effective include embryo transfer,^8,30,38 cross-fostering,^2,5,33,36,41 treatment of individual mice with antibiotics,^9,10,26 and cross-fostering in combination with a medicated diet.¹⁸ In contrast to these methods, successful dietary treatment has the potential to be very useful for eradicating multiple Helicobacter spp. in large mouse colonies without the need for surgery or individual manipulation, particularly from colonies of genetically manipulated mice that are not available commercially and are expensive or difficult to rederive by existing methods. However until recently, attempts to eliminate Helicobacter spp. by using dietary treatment alone have been largely unsuccessful. Eradication of Helicobacter spp. was not achieved in scid/Trp53 knockout mice³² or TCR × Rag, HNT/TCR BALB/c, and TNF transgenic mice¹⁸ by using a diet containing amoxicillin, metronidazole, and bismuth or in B6.129P2-IL10^tm1Cgn/J mice³¹ by using a dietary treatment with amoxicillin, clarithromycin, metronidazole, and omeprazole. Successful eradication of Helicobacter spp. by using this same 4-drug combination diet has been reported in rats¹⁷ and mice with a musculoskeletal deficiency but no known immune deficiency,¹⁹ although the infection status of individual mice in that study was not determined before treatment. Preliminary information from our institution suggests that this 4-drug diet was effective in eradicating Helicobacter spp. in mice deficient in functional natural killer cells. Therefore, the current prospective controlled study was undertaken to evaluate the effectiveness of the 4-drug medicated diet in eradicating H. hepaticus, H. bilis, and H. rodentium from 2 naturally infected strains of immunocompromised mice.     

Resistance of Sprague–Dawley Rats to Infection with Helicobacter pullorum

Helicobacter pullorum is an enterohepatic Helicobacter spp. known to infect both chickens and humans. H. pullorum infection has recently been reported in both mice and rats. This study was designed to determine whether standard methods of colony health surveillance using exposure to rodent soiled bedding could detect H. pullorum in Sprague–Dawley rats. We exposed 8 Helicobacter-free Sprague–Dawley rats to bedding from H. pullorum-infected Brown Norway rats. Fecal samples were analyzed by PCR every 2 wk for 22 wk. Dirty bedding transfer resulted in intermittent positive fecal PCR results; however, none of the rats became persistently infected with H. pullorum. Select intestinal tissues collected at necropsy analyzed by PCR were negative for H. pullorum. To determine whether the failure to detect H. pullorum in Sprague–Dawley rats receiving contact bedding was due to resistance of Sprague–Dawley rats to H. pullorum colonization, 10 Helicobacter-free Sprague–Dawley rats were orally dosed with H. pullorum. Fecal samples were analyzed by PCR every 2 wk for 12 wk. At 2 wk after infection, 5 of 10 rats were PCR positive for H. pullorum. By 12 wpi, only 2 rats were persistently colonized with H. pullorum according to culture and PCR results. These data contrast with our previous data, which showed a high frequency of both natural and experimental H. pullorum infection in Brown Norway rats. Sprague–Dawley rats are resistant to experimentally induced H. pullorum gastrointestinal colonization when dosed orally with H. pullorum or exposed to bedding from H. pullorum-infected rats.Abbreviations: wpi, weeks postinfection; EHS, enterohepatic Helicobacter speciesThe genus Helicobacter is generally separated into 2 groups—gastric Helicobacter species and enterohepatic Helicobacter species (EHS)—dependent on the preferred site of colonization. EHS preferentially colonize the gastrointestinal tract and, in some cases, the biliary tree of their host. In 1994, the novel Helicobacter species, H. pullorum, was isolated from human feces and the intestinal contents and livers of chickens.²¹ Subsequent to its original isolation and characterization, H. pullorum has been reported to infect humans, chickens, turkeys, guinea fowl, mice, and (most recently) Brown Norway (BN/MolTac) rats (Rattus norvegicus).^2,4,16,21,24H. pullorum is associated most often with farm-raised poultry and is suspected to cause vibrionic hepatitis in chickens.^16,21,28,29 In humans, infection with H. pullorum has been associated with diarrhea. In addition, PCR assays have identified this organism in patients with inflammatory bowel disease, hepatitis, cholecystitis, and hepatocellular carcinoma.^{3,5,9,12,18,22,25}We recently reported that Brown Norway rats are highly susceptible to both natural and experimental H. pullorum infection.⁴ Another study that reported colonization of H. pullorum in rats detected the organism in intestinal contents by using denaturing gradient gel electrophoresis after rats were treated with a common carcinogen (4-nitroquinoline-1-Oxide). Clinical signs ascribed to H. pullorum infection were not noted.³⁰Transmission of EHS is known to occur through the transfer of dirty bedding in mice.^14,23,26 Sprague–Dawley (Crl:SD) rats are commonly used in surveillance programs as sentinels to ascertain disease status within the colony, and the use of dirty-bedding sentinels is a common practice in surveillance programs. The purpose of the current study was to ascertain the effectiveness of dirty bedding transfer in the detection of H. pullorum infection of rats. In addition, to explore their susceptibility to H. pullorum colonization, we orally dosed Sprague–Dawley rats with H. pullorum.     

Effects of Medicated Diet to Eradicate Helicobacter spp. on Growth,Pathology, and Infection Status in Rag1–/– and Nude Mice

The use of a commercial 4-drug diet has been shown to eradicate Helicobacter spp. from immunocompetent mice and those with innate immunodeficiencies. However the efficacy of this diet has not been confirmed in mice with altered adaptive immunity. We hypothesized that an 8-wk treatment with medicated diet would eradicate H. hepaticus and H. typhlonius from young naturally infected nude and Rag1 mice lacking functional T cells (Foxn1^nu) or T and B cells (B6.129S7-Rag1^tm1Mom/J), respectively. We evaluated helicobacter status, body weight, and gross and histologic changes between medicated and control diet in groups of infected and uninfected mice throughout treatment and at 8 wk after treatment completion. Initial infection status was confirmed by fecal PCR at weaning and 3 wk later, with study initiation in 7-wk-old mice. PCR testing demonstrated that independent of strain and sex, all treated mice tested negative for Helicobacter spp. after 4 wk of treatment and remained negative for the duration of the study. Irrespective of infection status, nude and Rag1 mice fed 8 wk of medicated diet gained less weight than did their untreated controls. Both strains normalized body weight while on control diet for the 8 wk after treatment. Mice fed medicated diet developed severe gastroesophageal hyperkeratosis, suggestive of reduced feed consumption, and enlarged ceca. These conditions improved or resolved after the return to control diet. This report is the first to demonstrate the efficacy and physical effects of providing medicated diet for the eradication of Helicobacter spp. from mice with adaptive immune deficiencies.Abbreviation: IBD, inflammatory bowel disease; NU/J, Foxn1^nu1; Rag1^tm1Mom, B6.129S7- Rag1^tm1Mom/JDespite 2 decades of reports documenting Helicobacter-associated gastrointestinal disease in mice, infections continue to persist widely not only at academic institutions in the United States but also at commercial vendors in other regions of the world.^1,3,31,44 Within the academic setting, health monitoring and exclusion policies vary markedly between universities and even within different animal facilities at a single institution. Often these policies are based on financial factors (the costs of screening by PCR and the resources required to rederive infected animals) in addition to the potential for Helicobacter infections to confound research.Attempting to predict the overall effect of infection on research can also be problematic. The development and severity of gastrointestinal pathology can vary considerably by mouse strain, species of Helicobacter, and disease model. Some strains of mice, including A/JCr, BALB/cAnNCr, C3H/HeNCr, and SJL/NCr, are particularly susceptible and develop chronic enterohepatic disease of considerable severity.^{19,21,30,49,50} In addition, several immunodeficient strains of mice develop severe disease after chronic infection. C.B-17/Icr-Prkdc^scid (SCID/NCr) mice, which lack functional T and B cells, develop progressive hepatitis and proliferative typhlocolitis after natural infection with H. hepaticus.³⁰ IL10^−/− mice such as B6.129P2-IL-10^tm1Cgn/J develop severe typhlocolitis after infection with several Helicobacter spp.^51,52 In contrast to immunocompetent strains, immunodeficient mice may manifest clinical signs such as diarrhea, perianal bleeding and rectal prolapse of variable severity.^18,29,51The species of Helicobacter affects the severity of disease. Although H. hepaticus remains the most well-studied enterohepatic mouse species, other closely related Helicobacter spp. also result in gastrointestinal disease.^15,31 Natural and experimental monoinfection with H. typhlonius led to typhlocolitis in C57BL/6J IL-10^−/− and SCID/NCr mice.^18,22,23 H. mastomyrinus infection led to granulomatous typhlocolitis (inflammatory bowel disease, IBD) in telomerase-deficient C57BL/6J mice during crucial early-senescence studies.¹⁵ Interestingly, gastrointestinal disease was significantly more severe in mice infected with H. mastomyrinus than in those infected with H. hepaticus.¹⁵In addition, the research impact of Helicobacter infections varies with disease model in a complex dynamic resulting from interactions between host gastrointestinal immunity, microflora, diet, and environmental conditions. Mouse models of IBD highlight these complexities. Helicobacter spp. infection rather than genetic modification was found to be responsible for the susceptibility and pattern of IBD development in T cell receptor αβ mutant mice.^6,10 Intentional inoculation has been used to study Helicobacter spp.- associated alterations in resident intestinal microflora and induction and severity of IBD in immunodeficient mice.⁵¹Given the difficulties of predicting the research impact of Helicobacter spp. infection in mice, perhaps tolerance for enzootic infections should be reconsidered. Benefits to eradication include not only elimination of the agent as an experimental confounder but also as a means to improve welfare through reduced clinical disease.¹ Complete exclusion of infected animals may serve as the least labor intensive and most cost effective strategy. A 10-y institution-wide exclusion policy that required all imported mice to be either rederived by embryo transfer or purchased from an approved Helicobacter-free vendor resulted in either complete elimination or significant reduction in 4 facilities tested in 1999 and again in 2009.³¹ Although effective, this method has the potential to interfere with the ability of individual investigators to receive mice from collaborating institutions that maintain facilities of unknown or positive infection status. In addition, this strategy would be ineffective for inhouse breeding colonies of genetically modified mice. In such cases, rederivation by in vitro fertilization, embryo transfer, and postpartum cross-fostering have all been proven successful. Although often effective, embryo transfer can be impeded by factors such as insufficient response to superovulation, unavailability of stud males, inadequate yield of fertilized eggs, unsuccessful embryo transfer, and the need for genotyping—all of which delay the initiation of research.⁴⁷ Furthermore, the detection of H. typhlonius from sex organs of both female and male Hsd:Athymic Nude-Foxn1^nu mice has demonstrated the potential for transmission from vasectomized male and recipient female mice.³⁸Rederivation by cross fostering has been used to eliminate enzootic Helicobacter spp. infections.^1,46 This technique is less costly and labor intensive and requires less expertise than embryo transfer.^1,42,46 One study comparing 2 cross fostering paradigms to eliminate Helicobacter spp., murine norovirus, mouse hepatitis virus and Syphacia obvelata found success was dependent on both pup age at the time of transfer and bedding changing patterns.¹ Pups transferred within 24 h of birth from cages that underwent bedding changes every 24 h tested negative more frequently than did pups transferred within 48 h of birth from cages containing up to 7-d-old dirty bedding.¹ However neither paradigm was completely successful in Helicobacter elimination, as evidenced by follow-up PCR testing of cross-fostered offspring.¹ Furthermore, isolation of H. hepaticus from the viscera of several late-stage C.B-17/Icr-Prkdc^scid (SCID/NCr) embryos belonging to an infected dam suggests that transplacental transmission is possible in immunodeficient mice.³⁰The administration of antibiotics to eliminate Helicobacter spp. in rodents has evolved considerably since first used to prevent chronic active hepatitis and colitis in young male SCID/NCr mice naturally infected with H. hepaticus.³⁶ In that initial report, various combinations of amoxicillin, bismuth, metronidazole, neomycin, and tetracycline were either added to the drinking water or administered orally 3 times daily for 14 d.³⁶ Although clearly labor-intensive, the cited study³⁶ determined that H. hepaticus could be eliminated by using all treatment regimens that included amoxicillin. A similar dosing regimen using either amoxicillin or tetracycline-based triple therapy was used to eliminate H. hepaticus from infected A/JCr mice.¹⁶ The first medicated diet formulation intended to eliminate infection in mice was a triple therapy of amoxicillin, metronidazole, and bismuth.¹⁷ When fed continuously for 2 wk to 6- to 10-mo-old DBA/J mice, the diet successfully eliminated chronic H. hepaticus infection as confirmed by posttreatment culture and PCR testing 1 mo later.¹⁷ Despite this success, the same amoxicillin-based triple combination diet fed for an unspecified duration failed to eliminate H. hepaticus from an immunodeficient breeding colony of Rag1 ^−/− mice.^16,51 When administered to a SCID (Prkc^scid/Tpr53^tm1tyi on a B6.129/Sv × C.B17 background) breeding colony severely affected by diarrhea associated with H. rodentium and H. bilis coinfection, the triple therapy reduced clinical illness during treatment period but did not eliminate infection or persistent diarrhea.⁴¹Although a newer medicated diet containing amoxicillin, clarithromycin, metronidazole, and omeprazole has been available for a decade, reports of efficacy are still quite limited. One study demonstrated successful eradication of H. hepaticus and H. bilis from a colony of 129 × 1/SvJ desmin-null and heterozygotic mice after 8 wk of continual treatment and 19 mo of PCR follow-up testing.²⁸ In addition, the 4-drug therapy was successful in eliminating Helicobacter spp. from several genetically modified rat strains whereby infected male rats were medicated for 3-two week cycles and pregnant rat dams and offspring were fed continuously from day 7 of gestation through weaning.²⁶ Posttreatment follow-up testing for 8 mo by fecal PCR confirmed that all treated rats remained negative.²⁶ More recently, we have reported the successful eradication of H. hepaticus, H. bilis, and H. rodentium from 2 strains of mice with innate immune deficiencies.⁹Questions remain, however, regarding the broad applicability of medicated diet for the elimination of Helicobacter spp., particularly with regard to strains with modifications of the immune system. The 4-drug medicated diet was unable to eradicate Helicobacter spp. from in B6.129P2-IL10^tm1Cgn/J mice,⁴⁰ and there is no information regarding efficacy in mice with deficiencies in adaptive immunity. Given the widespread use of these mice in gastrointestinal cancer and IBD research, evaluation of medicated diet to eradicate Helicobacter spp. from such strains is warranted. Although many immunodeficient inbred strains are available Helicobacter-free from commercial vendors, these strains often are genetically modified further, maintained, and imported from collaborating institutions with endemic Helicobacter spp. infections that need to be eliminated in a timely manner. We therefore conducted a prospective controlled study to evaluate the potential of the 4-drug medicated diet to eradicate H. hepaticus and H. typhlonius from young, naturally infected nude and Rag1 mice lacking functional T cells (Foxn1^nu) or T and B cells (B6.129S7-Rag1^tm1Mom/J), respectively. We also evaluated the physical effect of both Helicobacter infection and medicated diet in growing mice by weekly recordings of body weight and assessment of gross and histologic changes between medicated and control diet in groups of infected and uninfected mice after an 8-wk treatment course and again 8 wk after treatment completion.     

STUDIES ON BLOOD FACTORS RhA, RhB, AND RhC

Observations are described of the incidence among Caucasians and Negroes of the blood factors Rh^A, Rh^B and Rh^C which occur associated with the Rh₀ factor in typical Rh-positive blood. The antiserums used for the tests were derived from Rh-positive patients who had had hemolytic transfusion reactions or erythroblastotic babies. Among a large series of individuals, it was found that only rarely is any of the blood factors Rh^A, Rh^B, or Rh^C lacking from "standard" Rh₀-positive blood. On the other hand, about half of the specimens of Rh₀ variant blood lack one or more of the factors Rh^A, Rh^B, and Rh^C, which, when present in such blood, are also almost always variants. Judging from the incidence of specimens lacking one or more of these factors, Rh^A, Rh^B, and Rh^B appear to be relatively independent of one another despite their association with blood factor Rh₀. Tests for factors Rh^A, Rh^B, and Rh^C distinguish new rare varieties of Rh and h agglutinogens, each genetically determined by corresponding allelic genes. There is no doubt that more clinical cases will be found in which sensitized Rh-positive individuals have antibodies resembling anti-Rh₀ in specificity. Four such cases have already been studied by the present authors, and in each case the antibodies were shown to be different from anti-Rh₀ in specificity. Since they were also different from one another, they have been assigned the symbols anti-Rh^A, anti-Rh^B, anti-Rh^C, and anti-Rh^D, respectively, the first three being the antiserums used in the present study. Obviously, in order to avoid confusion of nomenclature, the specificity of antiserums from other similar cases will have to be compared with anti-Rh^A, anti-Rh^B, anti-Rh^C, and anti-Rh^D and shown to be different from all four, as well as anti-Rh₀, before a distinctive symbol is assigned to them.     

Helicobacter spp. in Wild Mice (Peromyscus leucopus) Found in Laboratory Animal Facilities

Wild rodents are a potential source for pathogen introduction into laboratory animal research facilities. A study was designed to assess wild mice found at our institution by infectious disease surveillance. Wild white-footed mice (Peromyscus leucopus) were captured with live capture traps placed in areas in which wild mice had been reported in several animal facilities. Captured animals were euthanized by inhalation of CO₂, blood was collected by cardiocentesis (n = 10), and necropsy was performed (n = 8). Serum samples were negative for antibodies to mouse parvovirus (types 1 and 2), mouse minute virus, Sendai virus, pneumonia virus of mice, mouse hepatitis virus, Theiler murine encephalomyelitis virus, reovirus, rotavirus, lymphocytic choriomeningitis virus, mouse adenovirus, ectromelia virus, K virus, cilia-associated respiratory bacillus, and Mycoplasma pulmonis. Of the 8 animals that were necropsied, pelt and cecal examinations were negative for ectoparasites and pinworms, respectively. Histopathologic examination of brain, heart, lungs, liver, kidney, spleen, stomach, and small intestine revealed bacteria morphologically compatible with Helicobacter spp. in the cecal and colonic glands and occasionally in the gastric lumen and pits. Mesenteric lymph nodes and feces from 8 of the animals were submitted for PCR analysis for the detection of mouse parvovirus, mouse minute virus, mouse hepatitis virus, and Helicobacter spp.; 7 of the samples were PCR-positive for Helicobacter spp. At this time, wild mice found in our animal facilities do not appear to be a significant source of common laboratory mouse viral pathogens. However, they are a potential source of Helicobacter infections.Despite careful efforts to prevent invasion of wild mice into research facilities, these pests sometimes gain access to areas that house or use laboratory animals. This risk is increased during the building of new animal facilities, when portions of the structure are open to the environment during construction. In addition to perpetuating physical damage to facilities by chewing, feeding, nest building, and contamination with urine and feces, these wild rodents represent a potential source of infectious agents to laboratory rodents. When laboratory rodents are housed in microisolation caging or ventilated caging systems, agents of particular concern would be those that are persistent in the environment, such as the murine parvoviruses. These types of organisms can be present on surfaces and materials that may come into contact with laboratory rodents, including work and cage surfaces. Rodents housed in conventional caging (with open, wire cage tops) have an increased chance of exposure to any infectious agent that wild rodent pests may carry because the laboratory and wild animals could interact directly with one another and because laboratory rodents could be exposed to feces and urine from wild rodents.Published reports regarding assessment of wild mice for the presence of infectious diseases of concern to laboratory rodents are sporadic. Deer mice (Peromyscus maniculatus) found in New Mexico were free of bacterial, viral, and parasitic pathogens.³ Wild house mice (Mus musculus) captured in Idaho and 2 tropical pacific islands and imported to start breeding colonies were reported to have serum antibodies to murine cytomegalovirus, mouse hepatitis virus, and lymphocytic choriomeningitis virus.⁷ Wild Mus musculus captured around the London zoo were reported to be infected with the pinworm species Aspiculuris tetraptera,² and other studies have found Helicobacter species in wild mice captured in forests in Brazil³ and the around an urban university in the United States.⁹In response to reports of wild mice in some of our animal facilities, a program of live trapping, necropsy, and infectious-agent screening was instituted. Wild white-footed mice (Peromyscus leucopus; also known as the wood mouse) were captured in live traps and euthanized for assessment. This species is a grayish or brownish rodent found over a large geographic area, ranging from Canada to Central America, is semiarboreal and omnivorous, and inhabits brushy and woody habitats. In addition, P. leucopus can also be found year-round in human-occupied buildings.⁵To date only 10 wild mice have been captured in our facilities, 6 of which were captured in a new facility that was in the final stages of construction. Captured mice were assessed for evidence of common rodent pathogens according to the protocols used for routine rodent health surveillance in our institution.     

Physicochemical,morpho-structural,and biological characterization of polysaccharides from three Polygonatum spp

Polygonatum species, including P. cyrtonema, P. kingianum, and P. sibiricum, are edible plants with medicinal purposes, which have long been consumed as food due to their high nutritional value. In this study, polysaccharides from P. cyrtonema (PCP), P. kingianum (PKP) and P. sibiricum (PSP) were obtained, and their physicochemical properties and in vitro biological activities were investigated. Our results demonstrated that PCP, PKP, and PSP consist of major fructose and minor glucose, galacturonic acid, and galactose in different molar ratios with the molecular weights of 8.5 × 10³ Da, 8.7 × 10³ Da, and 1.0 × 10⁴ Da, respectively. The three polysaccharides had triple-helical structures with β-d-Fruf, α-d-Glcp, α-d-Galp sugar residues, and an O-acetyl group, and displayed peak-shaped structures in different sizes. They also exhibited thermal, shear-thinning behavior and viscoelastic properties, and PCP presented the highest viscoelasticity. Moreover, they exerted strong free radical-scavenging abilities, and significant reducing capacity. PCP was the strongest, followed by PSP and then PKP. They significantly promoted the polarization of the M1 macrophage, with the effect of PCP ranking first. All three had similar effects on GLP-1 secretion. It is, therefore, necessary to identify the various roles of these three Polygonatum polysaccharides as functional agents based on their bioactivities and physicochemical properties.

Three Polygonatum polysaccharides with different physicochemical properties exert distinct effects on free radical-scavenging abilities and the promotion of M1 macrophage polarization, while they have similar effects on GLP-1 secretion.     

Prevalence of Murine Helicobacter spp. Infection Is Reduced by Restocking Research Colonies with Helicobacter-Free Mice

Most academic research colonies of mice are endemically infected with enterohepatic Helicobacter spp. (EHS). We evaluated EHS prevalence in surveillance mice before and after a 10-y period of requiring that imported mice be free of EHS by embryo transfer rederivation or purchase from approved vendors. In 2009, composite fecal samples from CD1 surveillance mice representing colony health in 57 rooms located in 6 facilities were evaluated for EHS infection by using PCR assays. Fecal samples were screened with primers designed to detect all known EHS, and positive samples were further assayed by using primers specific for H. hepaticus, H. bilis, H. rodentium, and H. typhlonicus. Most EHS were detected in surveillance mice within the first month of dirty bedding exposure, with prevalence ranging from 0% to 64% as monoinfections or, more commonly, infections with multiple EHS. Compared with 1999 prevalence data, EHS remained endemic in colonies importing the lowest number of EHS-free mice. EHS were absent or the prevalence was greatly reduced in colonies receiving the highest percentage of EHS-free mice. This study demonstrates that the management decision to require exclusive importation of EHS-free mice reduced EHS prevalence on an institutional scale without intensive labor and expense associated with other techniques or interference with research objectives.Abbreviation: EHS, enterohepatic Helicobacter spp.; ET, embryo transfer; Hb, H. bilis; Hh, H. hepaticus; Hm, H. mastomyrinus; Hr, H. rodentium; Ht, H. typhlonicusEnterohepatic Helicobacter spp. (EHS) infections are endemic in the majority of research mouse colonies. In 2007, 84% of mice shipped from academic institutions worldwide for embryo transfer (ET) rederivation at our institution were PCR-positive for EHS. H. hepaticus (Hh) was detected in 64% of the mouse shipments either as a monoinfection or in combination with other EHS including H. bilis (Hb), H. rodentium (Hr), H. typhlonicus (Ht), and H. mastomyrinus (Hm).³⁰ Although EHS generally cause subclinical infection in immunocompetent mice, opportunistic infections have the potential to confound experimental data in mouse models.^9,17,34 Importantly, chronic EHS infection in immunodeficient and select inbred strains of mice can induce liver¹⁰ and lower bowel carcinoma,¹³ typhlocolitis, and rectal prolapse,^16,21,28 and reduce reproductive performance.²⁵ In addition, EHS-induced inflammatory responses may alter host immune responses to unrelated experimental infections (for example, promoting elevated systemic IFNγ responses).^3,20Key challenges to eradication of EHS from rodent colonies are determining infection status, eliminating endemic infections, and instituting management practices that prevent reinfection. EHS are disseminated through fecal–oral transmission within a colony and are transmissible to surveillance mice through dirty-bedding exposure.^1,19,24,32 For routine surveillance, PCR assay of feces or cecal mucosal scrapings for genus-specific Helicobacter 16S rRNA genes is the most efficient means of detecting EHS infection, with speciation (if desired) of positive results by culture, restriction fragment length polymorphism analysis, species-specific PCR, or sequence analysis.³⁴ In 1999, as determined by species-specific PCR assays of cecal scrapings from 59 surveillance mice exposed to dirty bedding from colony mice in 26 rooms representing 4 mouse facilities, EHS were endemic on our campus, with prevalence in surveillance mice of 41% for Hh, 82% for Hr, and 6% for Hb.³² Husbandry practices used to minimize cage-to-cage transmission of EHS included microisolation caging, sanitized forceps to transfer mice, and a cage change order from known Helicobacter-free mice to mice of unknown or known EHS infection status (that is, clean to dirty traffic flow of personnel and equipment).³² Although EHS eradication potentially could be accomplished campus-wide by using labor-intensive antibiotics^7,15 and cross-fostering,^4,29,31 we hypothesized that a more cost-effective approach, without confounding experimental data, would be to restrict importation of mice to EHS-free sources. Vendors were screened to establish that production colonies were SPF for EHS, and a new requirement was instituted for embryo transfer (ET) rederivation of mice obtained from random sources, typically other academic institutions, replacing traditional quarantine practices. This study used PCR data from 1999 and 2009 to evaluate the success of this approach, which was defined as a marked decrease in the prevalence of EHS infection over time.     

Diagnosis of Aeromonas hydrophila, Mycobacterium species, and Batrachochytrium dendrobatidis in an African Clawed Frog (Xenopus laevis)

Here we describe diagnosis of concurrent infection with Aeromonas hydrophila, Mycobacterium spp., and Batrachochytrium dendrobatidis in a wild female Xenopus laevis captured in Chile and transported to the United States. After approximately 130 d in the laboratory, the frog was presented for dysecdysis and obtundation. After euthanasia, tissues were submitted for histopathologic evaluation and PCR analysis for B. dendrobatidis and Ranavirus. Clinically significant gross lesions included cutaneous ulcerations on the lip, right forelimb, and ventral chest. Microscopic findings included regionally extensive splenic necrosis, diffuse pneumonia, and fibrinous coelomitis all containing intralesional bacteria. PCR analysis yielded positive results for B. dendrobatidis only. Bacterial culture of the ulcerated skin and liver yielded A. hydrophila. Infection with Contracaecum spp. was diagnosed as an incidental finding. To our knowledge, this case is the first report of simultaneous infection with Aeromonas hydrophila, Mycobacterium spp., and Batrachochytrium dendrobatidis in a laboratory-maintained X. laevis captured from the wild.The African clawed frog, Xenopus laevis, is likely the most widely used amphibian research model.^22,30 This species is an aquatic anuran and is readily suited for the research environment due to year-round gametogenesis, brief generation time, longevity in captivity, and ability to adjust to various laboratory conditions.¹¹ Xenopus oocytes historically have been used in human pregnancy assays,²² and their recent popularity is attributable to their widespread use in cell and molecular biology research^22,26 and developmental toxicology investigations.^22,30 Because clawed frogs are present in virtually all vivaria supporting the aforementioned research endeavors,^22,30 accurate diagnosis of clinical conditions is paramount for the laboratory animal practitioner and other research personnel.Bacterial infections have long been a challenge to investigators using frogs for research.^4,6,9,13,15 Mycobacterium spp. are implicated frequently as the cause of anuran disease. Species of Mycobacterium isolated from X. laevis include M. marinum, M. chelonae, M. xenopi, and M. liflandii.¹⁰ Mycobacterium spp. are potential zoonotic pathogens and thereby raise additional concerns for laboratory personnel. Aeromonas hydrophila has been reported to cause one of the most devastating infectious diseases in laboratory amphibians.³ Historically called red-leg disease, A. hydrophila infections have been cited for widespread amphibian mortality in wild and captive populations.^13,22 Recent evidence suggests multiple pathogens may cause signs similar to A. hydrophila. As a result, epizootics attributed to red-leg disease prior to the 1990s may have been misdiagnosed and over-reported. Newly recognized pathogens with similar clinical presentations include ranaviruses and the chytrid fungus Batrachochytrium dendrobatidis.⁵ As evidence of their world-wide significance, both Ranavirus and B. dendrobatidis have recently been listed as diseases notifiable to the World Organisation for Animal Health.³⁶ Since the mid1990s, amphibian mass mortalities and population declines in the wild have coincided with the sudden appearance of chytridiomycosis and its etiologic agent B. dendrobatidis.^20,34,35 Although predominately a disease of wild amphibians, chytridiomycosis is also problematic in captive colonies.^{5,7,19,23,24,35} To date, a single report has described B. dendrobatidis infection in laboratory-maintained frogs (X. tropicalis and X. laevis).²³ Here we discuss diagnosis of concurrent infection with A. hydrophila, Mycobacterium spp., and B. dendrobatidis in a female X. laevis.     

Synthesis of novel antibacterial and antifungal quinoxaline derivatives

A series of quinoxaline derivatives were designed, synthesized and evaluated as antimicrobial agents against plant pathogenic bacteria and fungi. Some of these compounds exhibited significant antibacterial and antifungal activities in vitro. Compound 5k displayed good antibacterial activity against Acidovorax citrulli (Ac). Compounds 5j and 5t exhibited the most potent anti-RS (Rhizoctonia solani) activity, with the corresponding EC₅₀ values of 8.54 and 12.01 μg mL⁻¹, respectively, which are superior to that of the commercial azoxystrobin (26.17 μg mL⁻¹). Further, the scanning electron microscopy results proved that compound 5j had certain effects on the cell morphology of RS. Moreover, an in vivo bioassay also demonstrated that the anti-RS activity of compound 5j could effectively control rice sheath blight. These results indicate that quinoxaline derivatives could be promising agricultural bactericides and fungicides.

Structure of some commercial agents.     

Effect of Cage-Wash Temperature on the Removal of Infectious Agents from Caging and the Detection of Infectious Agents on the Filters of Animal Bedding-Disposal Cabinets by PCR Analysis

Efficient, effective cage decontamination and the detection of infection are important to sustainable biosecurity within animal facilities. This study compared the efficacy of cage washing at 110 and 180 °F on preventing pathogen transmission. Soiled cages from mice infected with mouse parvovirus (MPV) and mouse hepatitis virus (MHV) were washed at 110 or 180 °F or were not washed. Sentinels from washed cages did not seroconvert to either virus, whereas sentinels in unwashed cages seroconverted to both agents. Soiled cages from mice harboring MPV, Helicobacter spp., Mycoplasma pulmonis, Syphacia obvelata, and Myocoptes musculinus were washed at 110 or 180 °F or were not washed. Sentinels from washed cages remained pathogen-free, whereas most sentinels in unwashed cages became infected with MPV and S. obvelata. Therefore washing at 110 or 180 °F is sufficient to decontaminate caging and prevent pathogen transmission. We then assessed whether PCR analysis of debris from the bedding disposal cabinet detected pathogens at the facility level. Samples were collected from the prefilter before and after the disposal of bedding from cages housing mice infected with both MPV and MHV. All samples collected before bedding disposal were negative for parvovirus and MHV, and all samples collected afterward were positive for these agents. Furthermore, all samples obtained from the prefilter before the disposal of bedding from multiply infected mice were pathogen-negative, and all those collected afterward were positive for parvovirus, M. pulmonis, S. obvelata, and Myocoptes musculinus. Therefore the debris on the prefilter of bedding-disposal cabinets is useful for pathogen screening.Abbreviations: ABDC, animal bedding disposal cabinet; MAV, murine adenovirus K87; MHV, mouse hepatitis virus; MNV, murine norovirus; MPV, mouse parvovirus; MVM, minute virus of mice; MMBTU, million British thermal units; SW, Swiss WebsterThe use of evidence-based standard operating procedures in animal resource centers is crucial to cost containment and sustainable energy use. Understandably, biosecurity is a major driver of procedures and processes in rodent facilities and permeates virtually all aspects of animal resource operations, making it necessary to balance the cost: benefit of detection, prevention, and control of infection. As an example, recent publications that suggested that mouse parvovirus (MPV) infection can be caused by MPV-contaminated grains that were not inactivated during their processing into pelleted rodent feed^25,36,47 have led to changes in husbandry procedures including the use of autoclaved or irradiated food. Although the successful control and prevention of MPV infections in our facilities has been attributed to the practice of autoclaving mouse cages preassembled with bedding and food prior to their use in the facility, this procedure was not definitively proven to be the sole factor in MPV eradication.³⁰ Because wash centers are historically the largest utilities consumer in animal facilities,¹⁵ this practice of autoclaving cages prior to use is not only labor-intensive but also energy-intensive, and the labor and energy consumption is amplified in facilities that lack a bulk autoclave and in which the cages must be transported to be autoclaved. In addition, during outbreaks with MPV and other infectious agents, our long-standing standard operating procedure for soiled cages is to autoclave them to inactivate infectious agents prior to removing the soiled bedding and cage washing. This labor- and energy-intensive practice of using autoclaving to decontaminate cages has historically been justified because MPV is a nonenveloped virus that is highly stable in the environment and difficult to inactivate.^{5,6,16,27,38,42,49} Despite the environmental stability of the virus, infections with MPV are difficult to detect because the amount of virus shed is low; transmission can be inefficient, resulting in inconsistent seroconversion of mice housed in the same cage; and PCR analysis can detect low levels of MPV DNA in the feces of mice that are unable to transmit virus to contact sentinels.^4,30,31 We recently demonstrated that cage washing alone removed or inactivated MPV from 14 cages that had housed outbred mice acutely infected with MPV; these findings are not surprising when the inefficiencies of MPV transmission are considered.¹¹ These initial results challenge the cost:benefit ratio of autoclaving cages as a means to decontaminate them prior to cage washing during an MPV outbreak. Our findings with MPV led us to question whether cage washing alone might be effective for decontaminating cages after exposure to other infectious agents that are stable in the environment, even if they are shed for longer periods of time or at higher levels than is MPV.In our previous study,¹¹ we showed that cage washing was effective at preventing fomite-based transmission of MPV by cage components, and we postulated that water temperature and detergent type contribute to MPV decontamination either directly through inactivation of the virus and/or indirectly through effective mechanical removal of organic waste and residual virus from the cage. The Guide for the Care and Use of Laboratory Animals²⁴ states that “disinfection from the use of hot water alone is the result of the combined effect of temperature and the length of time at a given temperature” and that “effective disinfection can be achieved with wash and rinse water at 143–180 °F or more.” Theoretically, contact times to disinfect equipment at these temperatures would need to be 1800 s at 143 °F (61.7 °C) and 0.1 s at 180 °F (82.2 °C).⁴⁶ Contact times of 6 s or more at 168 to 180 °F (75.6 to 82.2 °C) killed 3 types of bacteria (Pseudomonas aeruginosa, Salmonella cholerasuis, and Staphylococcus aureus) in one study,⁴⁵ and contact times of 2 min or more at 160 °F (71.1 °C) killed 5 types of bacteria (Escherichia coli, Klebsiella pneumonia, Proteus mirabilis, Providencia rettgeri, and Staphylococcus epidermidis) in another.³⁹ However, these 2 studies were performed by using hot water in a test tube and, therefore, detergent and mechanical spraying action that occur within rack washers was not a factor.Traditionally, the temperature used for the wash and rinse water in our facilities has been 180 °F (82.2 °C) with a belt speed of 2 to 3 ft./min (0.6 to 0.9 m/min). We postulated that if the volume and force of the wash water, combined with detergents, consistently diluted or removed infectious agents to below the level necessary for the transmission of infection, then wash temperatures high enough to inactivate the agents would be unnecessary. Given the energy usage and infrastructure necessary to boost ‘domestic’ hot water to 180 °F, the purpose of this study was to compare the efficacy of cage washing by using the domestic hot-water temperature (110 °F [43.3 °C]) and the traditional steam-boosted wash temperature (180 °F [82.2 °C]) on preventing the transmission from contaminated caging of 3 of the most prevalent viral agents (MNV, MPV, MHV; prevalence, 32.6%,1.8%, and 1.6%, respectively ) and bacterial and parasitic agents (Helicobacter spp. and pinworms; prevalence,15.9% and 0.3%, respectively).³⁵Effective and efficient decontamination of caging goes hand-in-hand with effective and efficient detection of infection. Timely detection of infection is important to biosecurity, and environmental sampling is a promising adjunct to sentinel exposure programs for the early detection of infectious agents. PCR analysis of cage and rack components, including the outflow prefilter of ventilated racks, has been shown to be of use for several infectious agents including MPV, MHV, Helicobacter spp., and fur mites.^10,26,31 The ability to reliably detect infectious agents from a site where soiled bedding debris is aerosolized and concentrated might provide an efficient adjunct for infectious agent screening. In the current study, we determined whether monitoring by PCR analysis of dust and debris collected from the animal bedding disposal cabinet (ABDC) prefilter could be used as an efficient adjunct to sentinel programs to screen for contamination by infectious agents.     

Hematologic, Serologic, and Histologic Profile of Aged Siberian Hamsters (Phodopus sungorus)

Biologic samples from 18 (12 female, 6 male) Siberian hamsters (Phodopus sungorus) representing an aged colony (17 to 27 mo) were examined. Values for CBC and serum biochemical parameters were determined, and macroscopic and microscopic pathologic evaluations were performed. Blood urea nitrogen levels were significantly higher in male (54.2 ± 14 mg/dL) compared with female (35.3 ± 22 mg/dL) hamsters and correlated histologically with a higher incidence of chronic glomerulonephropathy in males (5 of 6 males; 0 of 12 females). All 18 hamsters had histologic evidence of follicular mite infestation. Half (6 of 12) of the female hamsters showed cystic rete ovarii. Other histologic findings included thymic or thyroid branchial cysts (3 of 18), focal enteritis (2 of 18), and single cases of hepatic hemangiosarcoma, renal adenoma, subcutaneous mast cell tumor, cutaneous sebaceous adenoma, cutaneous trichofolliculoma, squamous papilloma of the nonglandular stomach, epididymal cholesteatoma, pyometra, and pituitary craniopharyngeal cyst. This study is the first published report of hematologic and serum chemical values for any population of Siberian hamsters and the first published report showing a potential male predisposition for chronic progressive glomerulonephropathy and a potential female predisposition for cystic rete ovarii.The Siberian hamster (Phodopus sungorus) is a small rodent native to the steppes of Kazakhstan, Manchuria, and Northern China. Phodopus sungorus originally was considered to be a single species encompassing 2 subspecies, P. s. sungorus and P. s. campbelli. However, crossbreeding, behavioral, karyotypic, and physiologic data suggest that P. sungorus and P. campbelli are 2 distinct species.^34,35 Siberian hamsters are noted for having fur along the surface of their feet and a modest, attenuated tail, leading to their nickname of ‘striped hairy-footed hamster.’Although Siberian hamsters are a popular pocket pet, they also increasingly are found in the laboratory setting due to unique physiologic properties that facilitate specific animal models of disease and behavior. Siberian hamsters have been studied with regard to circadian rhythm,^37,38 photoperiod and its effect on reproduction,^11,29 torpor,³⁶ sleep,²² hair-coat growth,²⁷ thermogenesis,^12,16 and fat metabolism.^12,16 The general advantages of Siberian hamsters as a laboratory animal include their ease of handling, small size (which enables efficient use of housing space), and their short reproductive cycle (which is the most compressed reproductive cycle of any eutherian mammal²⁵). Their average lifespan is reported as ranging from 12 to 24 mo.^15,17The more commonly used Syrian hamster (Mesocricetus auratus) has been characterized extensively biochemically and hematologically, establishing reliable normal reference ranges for both sexes at various age ranges. However, few current sources^17,23,24 of similar detailed information regarding the biochemical and hematologic parameters for P. sungorusare available.Documentation of spontaneous pathologic conditions in P. sungorus is also scarce. Prior reports have shown a limited number of spontaneous conditions (both neoplastic and nonneoplastic) within the more broad group of ‘Russian’ or ‘Djungarian’ hamsters, demonstrating a relatively high incidence of mammary tumors^21,30 and as integumentary fibromas.³ Although there is a paucity of information regarding spontaneous infectious disease within this particular species, experimental inoculation of P. sungorus with Neospora caninum and Babesia microti suggests susceptibility to these parasites.^19,39Our goal in the current study was to establish reference ranges for aged (17 to 27 mo) Siberian hamsters by examining serum biochemical and hematologic values of male and female animals. In addition, we wished to catalog any gross or microscopic abnormalities present in this population.     

Salt bridges govern the structural heterogeneity of heme protein interactions and porphyrin networks: microperoxidase-11

In this work, a proteolytic digest of cytochrome c (microperoxidase 11, MP-11) was used as a model to study the structural aspects of heme protein interactions and porphyrin networks. The MP-11 structural heterogeneity was studied as a function of the starting pH (e.g., pH 3.1–6.1) and concentration (e.g., 1–50 μM) conditions and adduct coordination. Trapped ion mobility spectrometry coupled to mass spectrometry (TIMS-MS) showed the MP-11 structural dependence of the charge state distribution and molecular ion forms with the starting pH conditions. The singly charged (e.g., [M]⁺, [M − 2H + NH₄]⁺, [M − H + Na]⁺ and [M − H + K]⁺) and doubly charged (e.g., [M + H]²⁺, [M − H + NH₄]²⁺, [M + Na]²⁺ and [M + K]²⁺) molecular ion forms were observed for all solvent conditions, although the structural heterogeneity (e.g., number of mobility bands) significantly varied with the pH value and ion form. The MP-11 dimer formation as a model for heme-protein protein interactions showed that dimer formation is favored toward more neutral pH and favored when assisted by salt bridges (e.g., NH₄⁺, Na⁺ and K⁺vs. H⁺). Inspection of the dimer mobility profiles (2+ and 3+ charge states) showed a high degree of structural heterogeneity as a function of the solution pH and ion form; the observation of common mobility bands suggest that the different salt bridges can stabilize similar structural motifs. In addition, the salt bridge influence on the MP-11 dimer formations was measured using collision induced dissociation and showed a strong dependence with the type of salt bridge (i.e., a CE₅₀ of 10.0, 11.5, 11.8 and 13.0 eV was observed for [2M + H]³⁺, [2M − H + NH₄]³⁺, [2M + Na]³⁺ and [2M + K]³⁺, respectively). Measurements of the dimer equilibrium constant showed that the salt bridge interactions increase the binding strength of the dimeric species.

In this work, a proteolytic digest of cytochrome c (microperoxidase 11, MP-11) was used as a model to study the structural aspects of heme protein interactions and porphyrin networks.     

Molar Malocclusions in Pine Voles (Microtus pinetorum)

Here we describe 5 cases of molar malocclusions in adult pine voles (Microtus pinetorum) used for behavioral endocrinology studies. This species belongs to the subfamily Microtinae, which possess aradicular hypsodont molars. The abnormal molars identified caused apparent difficulty in mastication, resulting in poor body condition necessitating euthanasia. Postmortem examination of the oral cavity revealed grossly elongated mandibular and maxillary molars with abnormal wear at occlusal surfaces. This colony health problem was addressed successfully by adding autoclaved hardwood sticks to each cage as an enrichment tool.Rodents of the genus Microtus typically are used as animal models in behavioral, physiologic, and ecologic studies. The genus Microtus, family Cricetidae, subfamily Arvicolinae²⁰ or Microtinae,¹⁰ comprises voles, including the meadow vole (M. pennsylvanicus) and prairie vole (M. ochragaster). Pine voles (M. pinetorum), also known as woodland voles, are found throughout eastern North America, from southern Canada through northern Mexico.²⁵ They are monogamous and possess several associated behavioral traits, such as cooperative breeding, the formation of pair bonds, and biparental care.^{3,9,17,18,27,28,30} These features have ensured their continued value to researchers engaged in behavioral neuroscience and endocrinologic fields.Dental anomalies are relatively common problems in laboratory rodents and lagomorphs. These abnormalities most often manifest as malocclusions due to the continuously growing teeth of these species.⁵ These continuously growing teeth typically are maintained to an appropriate length through normal wear from gnawing behavior but can become maloccluded and overgrown if normal wear is prevented. The molars of rodents of the genus Microtus, unlike those of rats and mice, grow continuously. Consequently, rodents of the genus Microtus may experience molar malocclusions as well as incisor malocclusions. Here we describe molar malocclusions in a laboratory-housed breeding colony of pine voles.     

Lack of Negative Effects on Syrian Hamsters and Mongolian Gerbils Housed in the Same Secondary Enclosure

In cases where different species might be housed in the same room or secondary enclosure, the Guide for the Care and Use of Laboratory Animals recommends that the animals should be behaviorally compatible and have the same health status. Syrian hamsters and Mongolian gerbils, both desert-dwelling rodents, appear to be reasonable candidates for such a combination. This study was undertaken to evaluate whether housing hamsters and gerbils in the same secondary enclosure is an acceptable practice. Weanling and breeding-age hamsters and gerbils were housed in open-topped cages in an isolator for 5 mo; the isolator also contained with nude and haired mice, which acted as sentinels. Cages housing hamsters and gerbils were rotated between species, and dirty bedding was exchanged between species in an effort to transmit microorganisms. In addition, sentinel mice housed in the isolator were supplied with dirty bedding from both hamsters and gerbils. Neither species showed clinical signs of illness, the health status of neither the hamsters nor the gerbils changed significantly, and the sentinel mice acquired only 2 infectious organisms, a Helicobacter species and Staphylococcus aureus. Both hamsters and gerbils bred successfully when housed together in the same isolator, and no infanticide or mortality was seen. Breeding performance did not differ between isolator breeding and barrier breeding. This study supports the housing of hamsters and gerbils in the same secondary enclosure.Although the 8th edition of the Guide for the Care and Use of Laboratory Animals generally recommends physical separation of animals by species, it also mentions that housing different species together in the same secondary enclosure (usually defined as a housing room) may be acceptable when the species are similar in pathogen status and are behaviorally compatible.¹⁰ Syrian hamsters (Mesocricetus auratus) and Mongolian gerbils (Meriones unguiculatus), both desert-dwelling rodents, appear to be reasonable candidates for such a combination. Syrian (or golden) hamsters inhabit extensive burrow systems in the arid, rocky plains of Syria.⁵ The burrow systems provide protection from predation and climactic extremes as well as serve as storage sites for hoards of food.⁵ Syrian hamsters are solitary animals, with males and females coming together only to mate, and whereas they are nocturnal in the laboratory, these hamsters are diurnal in the wild.^5,6 In comparison, Mongolian gerbils inhabit semiarid, sandy-soiled areas on the Mongolian steppes.⁸ Like hamsters, gerbils dig extensive burrow systems, where they hoard food, shelter from predators, and avoid climate extremes.^1,2 Gerbils are diurnal or periodically active.^2,17 Unlike hamsters, gerbils live in social groups.²Charles River Laboratories breeds both gerbils and hamsters at its Kingston, NY, facility, as well as its facility near Lyon, France. According to Charles River''s sales figures, the use of both hamsters and gerbils in research has declined from historical levels in both the United States and Europe. These species have been housed in separate rooms in the New York facility, but animal care and husbandry tasks could be accomplished more efficiently by combining the animals into a single room. Hamsters and gerbils inhabit similar habitats in the wild, and the 2 species have compatible environmental parameters in captivity. Because the native habitats of these species are geographically distant from one another, the predation of one species by the other is unlikely, as is one species serving as a source of stress for the other. Although Charles River''s gerbils and hamsters have slightly different health profiles, whether agents that colonize one species infect the other and, if agents are transmitted, whether colonization leads to clinical disease are unknown. Housing the animals closely together in an isolator and evaluating their health, reproduction, and behavior enables the evaluation of potential effects on both species. We tested the hypothesis that housing and breeding hamsters and gerbils in the same secondary enclosure has no observable negative effect on either species.     

Hematologic and Serum Biochemical Values of 4 Species of Peromyscus Mice and Their Hybrids

Deer mice (Peromyscus maniculatus) and congeneric species are used in a wide variety of research applications, particularly studies of developmental, physiologic, and behavioral characteristics associated with habitat adaptation and speciation. Because peromyscine mice readily adapt to colony conditions, animals with traits of interest in the field are moved easily into the laboratory where they can be studied under controlled conditions. The purpose of this study was to determine the serum chemistry and hematologic parameters of 4 frequently used species from the Peromyscus Genetic Stock Center species (P. californicus, P. leucopus, P. maniculatus, and P. polionotus) and to determine quantitative differences in these parameters among species and between sexes. Triglyceride values were substantially higher in female compared with male mice in all 4 species. Similar cross-species differences in MCH were present. Overall there was considerable interspecific variation for most blood parameters, with little evidence for covariation of any 2 or more parameters. Because crosses of P. maniculatus and P. polionotus produce fertile offspring, segregation analyses can be applied to determine the genetic basis of any traits that differ between them, such as their 3.8- and 2.1-fold interspecific differences in cholesterol and triglyceride levels, respectively. The current data provide a set of baseline values useful for subsequent comparative studies of species experiencing different circumstances, whether due to natural variation or anthropogenic environmental degradation. To enable such comparisons, the raw data are downloadable from a site maintained by the Stock Center (http://ww2.biol.sc.edu/~peromyscus).Abbreviations: BW, P. maniculatus bairdii; IS, P. californicus insignis; LL, P. leucopus; PO, P. polionotus subgriseusCollectively peromyscine rodents are the most common, abundant, and speciose native North American mammals. Ranging from Alaska to Central America and from the Atlantic to the Pacific, they occur in a wide range of habitats, including sea-level wetlands, beaches, forests, prairies, and deserts and mountains of elevations to 14,000 ft.^23,24 As such, peromyscine rodents are uniquely positioned as models for studying the factors, genetic and otherwise, responsible for reproductive isolation and speciation. In addition, they are useful as models to study the factors enabling adaptive responses to changing environmental conditions, to other species, and to each other.^10,18,35 Peromyscus maniculatus (deer mice) and P. leucopus (white-footed mice) are the most familiar, widespread, and biologically best-known species. In addition, these particular species have drawn considerable public health interest owing to their roles as zoonotic reservoirs of infectious disease organisms,⁴¹ notably hantavirus (P. maniculatus)²⁹ and the Borrelia spp. causing Lyme disease (P. leucopus).²⁷Peromyscines are reared in animal colonies incorporating caging, feeding, and maintenance regimens used for laboratory mice.²¹ The stocks maintained by the Peromyscus Genetic Stock Center (http://stkctr.biol.sc.edu/) were all derived from wild-caught animals and bred by random mating to maintain genetic diversity. As such, they can be considered to be closely related genetically to their wild counterparts and possess allelic combinations maximizing the physiologic and behavioral traits undergirding the species’ habitat adaptation. This situation contrasts with the standard inbred strains of laboratory mice, which are amalgams of genes derived from 3 or 4 different Mus spp.^6,16 and which lack wild natural counterparts. Random admixture of genes from different species may provide a means of unmasking gene effects that would otherwise go unnoticed, such as those being studied in collaborative cross strains.⁸ Yet the results say little regarding the adaptive roles of such genes in wild populations and their corresponding phenotypes.In the current report we present hematologic and biochemical comparisons of 4 species maintained by the Stock Center to determine the degree that species- and sex-associated factors affect various blood parameters. The phylogenetic relatedness of the 4 species is illustrated in Figure 1. The species examined were P. californicus insignis (IS), P. leucopus (LL), P. maniculatus bairdii (BW), and P. polionotus subgriseus (PO). Two of these (BW and PO) are interfertile sister species, whose offspring manifest disparate dysgenetic phenotypes depending on the parental sex.^9,12,51 Thus, PO females mated with BW males generate offspring manifesting lethal overgrowth and which rarely develop to term. Offspring of the reciprocal cross (BW females × PO males) are viable but undersized at birth and into adulthood. We determined the hematologic and chemical parameters of the BW × PO hybrids and compared the results with those of the parental stocks.Open in a separate windowFigure 1.Evolutionary relationship and species designations (in parentheses) of the peromyscine species whose hematologic and serum biochemical parameters were profiled.We undertook this study for 3 primary reasons: 1) to establish baseline values for commonly used stocks maintained by the Stock Center, thereby rendering them more useful and complementing their completed genome sequences (currently being assembled); 2) to compare species- and sex-associated differences in such values; and 3) to assess basic inheritance patterns of differences between the PO and BW stocks, which differ in numerous characteristics, including partner fidelity (PO and IS are monogamous species^10,14,19), stress response and blood glucose homeostasis, repetitive behaviors, growth control, and various blood parameters documented in this study.     

Development of Cooperative Primer-Based Real-Time PCR Assays for the Detection of Plasmodium malariae and Plasmodium ovale

Plasmodium malariae and Plasmodium ovale are increasingly gaining public health attention as the global transmission of falciparum malaria is decreasing. However, the absence of reliable Plasmodium species-specific detection tools has hampered accurate diagnosis of these minor Plasmodium species. In this study, SYBR Green–based real-time PCR assays were developed for the detection of P. malariae and P. ovale using cooperative primers that significantly limit the formation and propagation of primers-dimers. Both the P. malariae and P. ovale cooperative primer-based assays had at least 10-fold lower detection limit compared with the corresponding conventional primer-based assays. More important, the cooperative primer-based assays were evaluated in a cross-sectional study using 560 samples obtained from two health facilities in Ghana. The prevalence rates of P. malariae and P. ovale among the combined study population were 18.6% (104/560) and 5.5% (31/560), respectively. Among the Plasmodium-positive cases, P. malariae and P. ovale mono-infections were 3.6% (18/499) and 1.0% (5/499), respectively, with the remaining being co-infections with Plasmodium falciparum. The study demonstrates the public health importance of including detection tools with lower detection limits in routine diagnosis and surveillance of nonfalciparum species. This will be necessary for comprehensively assessing the effectiveness of malaria interventions and control measures aimed toward global malaria elimination.

Human malaria is a life-threatening disease caused by five distinct Plasmodium species (namely, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, and Plasmodium knowlesi).¹ Among these species, P. falciparum and P. vivax are the most prevalent and cause the most severe forms of the disease.^2,3 However, with the general global reduction in falciparum malaria, significant attention has been drawn toward the minor Plasmodium species: P. malariae and P. ovale.^4,5 Although these less prevalent species are generally associated with benign malaria,^6,7 recent reports have implicated them in major disease burden, such as severe anemia, acute respiratory distress syndrome, and acute renal failure.^8,9 Therefore, the availability of reliable tools for timely and accurate diagnosis of nonfalciparum malaria is necessary to inform appropriate treatment and effective management.Current methods for malaria diagnosis include microscopy, rapid diagnostic tests, and nucleic acid–based amplification tests (NAATs).¹⁰ However, because of the morphologic similarities among Plasmodium species and the low parasite densities of P. malariae and P. ovale species in clinical isolates, both microscopy and rapid diagnostic tests remain unsatisfactory for routine detection of nonfalciparum Plasmodium species.^6,11,12 Efforts to address this diagnostic gap led to the development of highly sensitive and specific NAATs, including PCR and loop-mediated isothermal amplification.^12,13Several NAATs, involving the use of TaqMan probes and SYBR Green, have been developed for the detection of nonfalciparum species.14, 15, 16, 17 Although these NAATs have improved sensitivity, lower detection limits, and higher specificity compared with microscopy and rapid diagnostic tests,¹³ the formation and propagation of primers-dimers remains a major sensitivity and specificity limiting factor, especially at low target concentration.^18,19 Attempts to address primers-dimers over the years led to the development of cooperative primers, which is the first technology that simultaneously curbs primer-dimer formation and propagation.²⁰ The cooperative primers were shown to significantly limit primer-dimer formation and propagation up to 2.5 million-fold compared with the conventional primers.²⁰A cooperative primer-based real-time assay [real-time quantitative PCR (qPCR)] has been developed for the detection of P. falciparum, and the assay was shown to have lower detection limit relative to its corresponding conventional primer-based assay.²⁰ In this study, SYBR Green–based qPCR assays were developed for the detection of P. malariae and P. ovale using cooperative primers that target the 18S ribosomal rRNA genes.