Mice are widely used as small animal models for influenza infection and immunization studies because of their susceptibility to many strains of influenza, obvious clinical signs of infection, and ease of handling. Analgesia is rarely used in such studies even if nonstudy effects such as fight wounds, tail injuries, or severe dermatitis would otherwise justify it because of concerns that treatment might have confounding effects on primary study parameters such as the course of infection and/or the serological response to infection. However, analgesia for study-related or -unrelated effects may be desirable for animal welfare purposes. Opioids, such as extended-release buprenorphine, are well-characterized analgesics in mice and may have fewer immune-modulatory effects than other drug classes. In this study, BALB/c and DBA/2 mice were inoculated with influenza virus, and treatment groups received either no analgesics or 2 doses of extended-release buprenorphine 72 h apart. Clinical signs, mortality, and influenza-specific antibody responses were comparable in mice that did or did not receive buprenorphine. We therefore conclude that extended-release buprenorphine can be used to alleviate incidental pain during studies of influenza infection without altering the course of infection or the immune response.

Rabbits can develop anemia due to serial phlebotomy or secondary to induced disease states. This study evaluated theeffects of a single injection and three consecutive injections of erythropoietin in rabbits at 150 IU/kg and 1,000 IU/kg in orderto determine whether these dosages produce a sustained increase in hematocrit. Analysis of CBC and chemistry parametersshowed significant elevation in hematocrit one week after administration of 1,000 IU/kg erythropoietin for three consecutivedays. These results indicate that this dosage regimen can increase hematocrit in apparently healthy, nonanemic rabbits forone week.

Background Influenza viruses pose significant disease burdens through annual seasonal outbreaks and unpredictable pandemics. Existing influenza surveillance programs have relied heavily on reporting of medically attended influenza (MAI). Continuously monitoring cause-specific school absenteeism may identify local acceleration of seasonal influenza activity. The Oregon Child Absenteeism Due to Respiratory Disease Study (ORCHARDS; Oregon, WI) implements daily school-based monitoring of influenza-like illness–specific student absenteeism (a-ILI) in pre-kindergarten through grade 12 schools and assesses this approach for early detection of accelerated influenza and other respiratory pathogen transmission in schools and surrounding communities.Methods Starting in September 2014, ORCHARDS combined automated reporting of daily absenteeism within 6 schools and home visits to school children with acute respiratory infections (ARI). Demographic, epidemiological, and symptom data are collected along with respiratory specimens. Specimens are tested for influenza and other respiratory viruses. Household members can opt into a supplementary household transmission study. Community comparisons are possible using a pre-existing, long-standing, and highly effective influenza surveillance program, based on MAI at 5 primary care clinics in the same geographical area.Results Over the first 5 years, a-ILI occurred on 6,634 (0.20%) of 3,260,461 student school days. Viral pathogens were detected in 64.5% of the 1,728 children with ARI who received a home visit. Influenza was the most commonly detected virus, noted in 23.3% of ill students. Influenza (p<0.001) and adenovirus (P=0.004) were significantly associated with a-ILI.Conclusion ORCHARDS uses a community-based design to detect influenza trends over multiple seasons and to evaluate the utility of absenteeism for early detection of accelerated influenza and other respiratory pathogen transmission in schools and surrounding communities. Initial findings suggest the study design is succeeding in collecting appropriate data to achieve study objectives.Competing Interest StatementDr. Jonathan Temte has received financial and material support from Quidel Corporation. Dr. John Tamerius and Quidel Corporation have generously supplied RIDT analyzers and tests.Funding StatementDr. John Tamerius and Quidel Corporation have generously supplied RIDT analyzers and tests. This study has been supported by CDC through the cooperative agreement # 5U01CK000542-02-00. The findings and conclusions in this study are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:All components of this proposed study were reviewed and approved by the Human Subjects Committees of the Education Research IRB - Social and Behavioral Sciences IRB (initial approval on September 4, 2013) and the University of Wisconsin Health Sciences-IRB (initial approval on December 5, 2013, with additional approvals as the protocol expanded and modified). The study is in full compliance with the Health Insurance Portability and Accountability Act of 1996 (HIPAA), FERPA, and all other federally mandated human subjects regulations. The US Office of Management and Budget has approved all forms used in this study.All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a s atement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesThe datasets generated and/or analyzed during the current study are not publicly available because the study is ongoing, but may be available from the corresponding author on reasonable request.4k4-year-old pre-kindergartena-Iabsenteeism due to illnessa-ILIabsenteeism due to influenza-like illnessa-TOTall-cause absenteeismARIacute respiratory infectionCDCCenters for Disease Control and PreventionCoVcoefficient of variationFERPAFamily Educational Rights and Privacy ActFluinfluenzaFDAFood and Drug AdministrationHIPPAHealth Insurance Portability and Accountability ActILIinfluenza-like illnessITinformation technologyIRBinstitutional review boardMAImedically attended influenzaNPnasopharyngealORCHARDSOregon Child Absenteeism Due to Respiratory Disease StudyOSDOregon School DistrictOPoropharyngealPCRpolymerase chain reactionRIDTrapid influenza diagnostic testRedCapResearch Electronic Data CaptureRPPrespiratory pathogen panelR/Erhinovirus/enterovirusRPhuman RNAse PSISschool information systemW-IISPWisconsin Influenza Incidence Surveillance ProjectWIRWisconsin Immunization RegistryWSLHWisconsin State Laboratory of HygieneVTMviral transport medium

Melioidosis, a potentially fatal infectious disease of humans and animals, including nonhuman primates (NHPs), is caused by the high-consequence pathogen Burkholderia pseudomallei. This environmental bacterium is found in the soil and water of tropical regions, such as Southeast Asia, where melioidosis is endemic. The global movement of humans and animals can introduce B. pseudomallei into nonendemic regions of the United States, where environmental conditions could allow establishment of the organism. Approximately 60% of NHPs imported into the United States originate in countries considered endemic for melioidosis. To prevent the introduction of infectious agents to the United States, the Centers for Disease Control and Prevention (CDC) requires newly imported NHPs to be quarantined for at least 31 d, during which time their health is closely monitored. Most diseases of public health concern that are transmissible from imported NHPs have relatively short incubation periods that fall within the 31-d quarantine period. However, animals infected with B. pseudomallei may appear healthy for months to years before showing signs of illness, during which time they can shed the organism into the environment. Melioidosis presents diagnostic challenges because it causes nonspecific clinical signs, serologic screening can produce unreliable results, and culture isolates are often misidentified on rapid commercial testing systems. Here, we present a case of melioidosis in a cynomolgus macaque (Macaca fascicularis) that developed a subcutaneous abscess after importation from Cambodia to the United States. The bacterial isolate from the abscess was initially misidentified on a commercial test. This case emphasizes the possibility of melioidosis in NHPs imported from endemic countries and its associated diagnostic challenges. If melioidosis is suspected, diagnostic samples and culture isolates should be submitted to a laboratory in the CDC Laboratory Response Network for conclusive identification and characterization of the pathogen.

Platynosomosis is a parasitic disease caused by a trematode of the genus Platynosomum, a bile duct and gallbladder fluke that has been described in captive neotropical primates (New World primates; NWPs) and causes high morbidity and variable mortality. Although it is a major concern for ex-situ conservation of these animals, there are only a few studies of platynosomosis in free-ranging NWPs. Therefore, the aim of this study was to characterize platynosomosis in a free-ranging population of marmosets (Callithrix spp) from the Brazilian Atlantic Forest, focusing on the epidemiological and pathological aspects of the disease. A total of 1,001 marmosets were evaluated and on the basis of clinicoepidemiological data, histopathology, histochemistry and immunohistochemistry, we concluded that Platynosomum spp infection has a prevalence of 8.9% (confidence interval: 7.3-10.8%) in free-ranging marmosets, with a higher frequency in the Metropolitan Region of Rio de Janeiro. Infection was associated with fibrosing and proliferative cholangiohepatitis associated with biliary lithiasis (3.0% of cases) and secondary bacterial infections (14.6% of cases).

Interleukin (IL)-11, a multi-functional cytokine, contributes to numerous biological processes, including adipogenesis, hematopoiesis, and inflammation. Asthma, a respiratory disease, is notably characterized by reversible airway obstruction, persistent lung inflammation, and airway hyperresponsiveness (AHR). Nasal insufflation of IL-11 causes AHR in wild-type mice while lung inflammation induced by antigen sensitization and challenge, which mimics features of atopic asthma in humans, is attenuated in mice genetically deficient in IL-11 receptor subunit alpha-1 (IL-11Rα1-deficient mice), a transmembrane receptor that is required conjointly with glycoprotein 130 to transduce IL-11 signaling. Nevertheless, the contribution of IL-11Rα1 to characteristics of non-atopic asthma is unknown. Thus, based on the aforementioned observations, we hypothesized that genetic deficiency of IL-11Rα1 would attenuate lung inflammation and increases in airway responsiveness following acute inhalation exposure to ozone (O(3)), a criteria pollutant and non-atopic asthma stimulus. Accordingly, four- and/or twenty-four hours following cessation of exposure to filtered room air or O(3), we assessed lung inflammation and airway responsiveness in wild-type and IL-11Rα1-deficient mice. With the exception of bronchoalveolar lavage macrophages and adiponectin, which were significantly increased and decreased, respectively, in O(3)-exposed IL-11Rα1-deficient as compared to O(3)-exposed wild-type mice, no other genotype-related differences in lung inflammation indices that we quantified were observed in O(3)-exposed mice. However, airway responsiveness to acetyl-β-methylcholine chloride (methacholine) was significantly diminished in IL-11Rα1-deficient as compared to wild-type mice following O(3) exposure. In conclusion, these results demonstrate that IL-11Rα1 minimally contributes to lung inflammation but is required for maximal airway responsiveness to methacholine in a mouse model of non-atopic asthma.

A 27-year-old female white-faced saki (Pithecia pithecia) died following an onset of vomiting and ptyalism. Necropsy revealed lesions of suppurative ventriculitis, choroid plexitis, periventricular encephalitis and meningitis with intralesional gram-positive coccobacilli and paired rods. The saki also had suppurative to mononuclear hepatitis, mild intestinal crypt necrosis, proliferative glomerulonephritis, aortic arteriosclerosis, pulmonary interstitial fibrosis, chronic mild epicarditis, ovarian medullary arteriopathy and a focal superficial cerebral fibrotic nodule with surrounding chronic mixed cell inflammation. Listeria monocytogenes was cultured from liver and spinal cord. Intralesional Listeria bacteria were immunolabelled in brain sections and real-time polymerase chain reaction of brain tissue detected L. monocytogenes. Whole genome multilocus sequence typing characterized the cultured bacterial isolates as sequence type 6 and clonal complex 6. A database search for related clinical and food listerial outbreaks identified genetically related isolates but, because these isolates were more than 20 alleles distant from the saki isolates, they were not a related cluster. Reports of listeriosis in non-human primates are infrequent, and when infections do occur, they tend to be haematogenous with the propensity to cause meningoencephalitis. This saki likely ingested environmental L. monocytogenes, which resulted in disease that may have been facilitated by pre-existing co-morbidities and age. © 2022 Elsevier Ltd

Ferrets are the gold-standard model for influenza A virus (IAV) research due to their natural susceptibility to human and zoonotic IAV, comparable respiratory anatomy and physiology to humans, and development of clinical signs similar to those seen in infected people. Because the presence and progression of clinical signs can be useful in infectious disease research, uncertainty in how analgesics alter research outcomes or compromise characteristics of disease progression have outweighed the concern regarding animal discomfort from these symptoms. Nonetheless, the principles of animal research require consideration of refinements for this important model for IAV research. Opioids offer a possible refinement option that would not directly affect the inflammatory cascade involved in IAV infection. Mirroring pathogenicity studies that use ferrets, 12 ferrets were inoculated intranasally with the A(H3N2) IAV A/Panama/2007/1999 and divided into 3 treatment groups ( n = 4 each), of which 2 groups received buprenorphine treatments on different schedules and the third received a saline control. The duration and location of viral replication, lymphohematopoietic changes, and clinical signs were comparable across all groups at all time points. High quantities of infectious virus in nasal wash specimens were detected in ferrets from all groups through day 5 after inoculation, and peak viral titers from the upper respiratory tract did not differ between ferrets receiving buprenorphine treatments on either schedule. Compared with the saline group, ferrets receiving buprenorphine exhibited transient weight loss and pyrexia, but all groups ultimately achieved similar peaks in both of these measurements. Collectively, these findings support the continued evaluation of buprenorphine as a refinement for IAV-challenged ferrets.

BACKGROUND: Influenza viruses pose significant disease burdens through seasonal outbreaks and unpredictable pandemics. Existing surveillance programs rely heavily on reporting of medically attended influenza (MAI). Continuously monitoring cause-specific school absenteeism may identify local acceleration of seasonal influenza activity. The Oregon Child Absenteeism Due to Respiratory Disease Study (ORCHARDS; Oregon, WI) implements daily school-based monitoring of influenza-like illness-specific student absenteeism (a-ILI) in kindergarten through Grade 12 schools and assesses this approach for early detection of accelerated influenza and other respiratory pathogen transmission in schools and surrounding communities. METHODS: Starting in September 2014, ORCHARDS combines automated reporting of daily absenteeism within six schools and home visits to school children with acute respiratory infection (ARI). Demographic, epidemiological, and symptom data are collected along with respiratory specimens. Specimens are tested for influenza and other respiratory viruses. Household members can opt into a supplementary household transmission study. Community comparisons are possible using a pre-existing and highly effective influenza surveillance program, based on MAI at five family medicine clinics in the same geographical area. RESULTS: Over the first 5 years, a-ILI occurred on 6634 (0.20%) of 3,260,461 student school days. Viral pathogens were detected in 64.5% of 1728 children with ARI who received a home visit. Influenza was the most commonly detected virus, noted in 23.3% of ill students. CONCLUSION: ORCHARDS uses a community-based design to detect influenza trends over multiple seasons and to evaluate the utility of absenteeism for early detection of accelerated influenza and other respiratory pathogen transmission in schools and surrounding communities.

Cocirculation of varying influenza types, strains, and lineages allows coinfection and intra-season sequential infection, although a same-strain sequential infection has not been previously described. This case report describes the first known case of sequential laboratory-confirmed influenza A (H3N2) infections in a child within one season.

A 10-y-old pigtail macaque presented with a subcutaneous, soft-tissue mass overlying the right stifle joint. Here we describe the clinical case and histopathologic and immunohistochemical analysis of this lesion. This case represents the first published report of juxtaarticular myxoma in a pigtail macaque.

Leptospirosis is a zoonotic infection caused by pathogenic members of the genus Leptospira spp. Knowledge of the prevalent serovars and their maintenance hosts is essential to understand the disease. The aim of this study was to evaluate the ability of serology by the microscopic agglutination test (MAT) to predict the serogroups compared with results of identification of leptospires in Sao Paulo, Brazil. MAT correctly assigned the serogroup of the infecting isolate in 49/52 cases (94.23%). The serogroup Icterohaemorrhagiae was the predominant serogroup (88.46%). This study showed the usefulness of the MAT to correctly identify the infecting serogroup with a good overall agreement between the serologically-identified infecting serogroup and by identification of the isolate and can be used in epidemiological surveys in Sao Paulo. However, it should be complemented by the identification of Leptospira isolates.

AIM: To improve evidence for public health practice, the conduct of effectiveness studies by practitioners is needed and may be stimulated if knowledge that smaller than usual samples may provide the same reliability of intervention effect size as larger samples. MATERIALS & METHODS: We examined reliability of intervention effect using computerized simulations of 2000 hypothetical immunization effectiveness studies from an actual study population and by small (30 and 60) and larger (100 and 200) control groups compared with an intervention group of 200 participants. RESULTS & CONCLUSION: Across simulated studies, the mean intervention effect (14%) and effect sizes were equivalent regardless of control group size and equal to the actual study effect. These results are relevant for similarly designed and executed studies and indicate that studies with smaller control groups can generate valid and accurate evidence for effective public health practice in communities.

Identification of the select agent Burkholderia pseudomallei in macaques imported into the United States is rare. A purpose-bred, 4.5-y-old pigtail macaque (Macaca nemestrina) imported from Southeast Asia was received from a commercial vendor at our facility in March 2012. After the initial acclimation period of 5 to 7 d, physical examination of the macaque revealed a subcutaneous abscess that surrounded the right stifle joint. The wound was treated and resolved over 3 mo. In August 2012, 2 mo after the stifle joint wound resolved, the macaque exhibited neurologic clinical signs. Postmortem microbiologic analysis revealed that the macaque was infected with B. pseudomallei. This case report describes the clinical evaluation of a B. pseudomallei-infected macaque, management and care of the potentially exposed colony of animals, and protocols established for the animal care staff that worked with the infected macaque and potentially exposed colony. This article also provides relevant information on addressing matters related to regulatory issues and risk management of potentially exposed animals and animal care staff.

The current status of Bartonella studies in mainland China is reviewed including both laboratory and ecological data and limited clinical data. Detection and isolation of Bartonella species from arthropods, pets and small wild animals is commonplace; this includes a variety of known and emerging Bartonella pathogens. In contrast, the medical literature analyzed from 1980 to 2010 consists of 31 reports of only of cat scratch disease (CSD). Most cases are from the East and South-Eastern provinces, the most populated areas with best access to medical care. Disease typically is described as febrile illness with symptoms traditionally reported for CSD elsewhere in the world. Clinical observations and anamnesis are the primary bases for diagnosis, since specialized serologic and molecular diagnosis is not widely available. Seroprevalence of healthy populations determined using Bartonella henselae antigen varies from 9.6 to 19.6%. The apparent discordance postulated between possible environmental exposure to diverse Bartonella agents and restricted B. henselae case etiologies suggests a need to determine whether other Bartonella species may also be etiologic agents of human illness and emphasizes the importance of applying modern diagnostic tools widely in clinical practice in mainland China.

Respiratory syncytial virus (RSV) is the most common cause of serious lower respiratory illness in infants and young children worldwide, making it a high priority for development of strategies for prevention and treatment. RSV can cause repeat infections throughout life, with serious complications in elderly and immunocompromised patients. Previous studies indicate that the RSV G protein binds through a CX3C chemokine motif to the host chemokine receptor, CX3CR1, and modulates the inflammatory immune response. In the current study, we examined the contribution of CX3CR1 to the immune response to RSV infection in mice. CX3CR1-deficient mice showed an impaired innate immune response to RSV infection, characterized by substantially decreased NK1.1(+) natural killer, CD11b(+), and RB6-8C5(+) polymorphonuclear cell trafficking to the lung and reduced IFNgamma production compared with those in wildtype control mice. Leukocytes from CX3CR1-deficient mice were poorly chemotactic toward RSV G protein and CX3CL1. These results substantiate the importance of the RSV G CX3C-CX3CR1 interaction in the innate immune response to RSV infection.

PURPOSE: To examine associations between biomarkers of joint tissue metabolism and whole blood lead (Pb), separately for men and women in an African American and Caucasian population, which may reflect an underlying pathology. METHODS: Participants in the Johnston County Osteoarthritis Project Metals Exposure Sub-Study (329 men and 342 women) underwent assessment of whole blood Pb and biochemical biomarkers of joint tissue metabolism. Urinary cross-linked N telopeptide of type I collagen (uNTX-I) and C-telopeptide fragments of type II collagen (uCTX-II), serum cleavage neoepitope of type II collagen (C2C), serum type II procollagen synthesis C-propeptide (CPII), and serum hyaluronic acid (HA) were measured using commercially available kits; the ratio of [C2C:CPII] was calculated. Serum cartilage oligomeric matrix protein (COMP) was measured by an in-house assay. Multiple linear regression models were used to examine associations between continuous blood Pb and biomarker outcomes, adjusted for age, race, current smoking status, and body mass index. Results are reported as estimated change in biomarker level for a 5-unit change in Pb level. RESULTS: The median Pb level among men and women was 2.2 and 1.9mug/dL, respectively. Correlations were noted between Pb levels and the biomarkers uNTX-I, uCTX-II, and COMP in women, and between Pb and uCTX-II, COMP, CPII, and the ratio [C2C:CPII] in men. In adjusted models among women, a 5-unit increase in blood Pb level was associated with a 28% increase in uCTX-II and a 45% increase in uNTX-I levels (uCTX-II: 1.28 [95% CI: 1.04-1.58], uNTX-I: 1.45 [95% CI:1.21-1.74]). Among men, levels of Pb and COMP showed a borderline positive association (8% increase in COMP for a 5-unit change in Pb: 1.08 [95% CI: 1.00-1.18]); no other associations were significant after adjustment. CONCLUSIONS: Based upon known biomarker origins, the novel associations between blood Pb and biomarkers appear to be primarily reflective of relationships to bone and calcified cartilage turnover among women and cartilage metabolism among men, suggesting a potential gender-specific effect of Pb on joint tissue metabolism that may be relevant to osteoarthritis.

It has been speculated that ticks may serve as vectors of Bartonella species. Circumstantial, clinical, epidemiological and serological evidence suggest that B. vinsonii subspecies berkhoffii (B. v. berkhoffii) might be transmitted by Rhipicephalus sanguineus. The purpose of the present study was to determine whether adult R. sanguineus ticks can be infected with a B. v. berkhoffii genotype II isolate via capillary tube feeding and whether the infection can then be transmitted from adult females to their eggs via trans-ovarial transmission. Furthermore, tick fecal material was also collected and screened as a possible source of infectious inoculum for canine infections. B. v. berkhoffii DNA was detected in 50% (7 of 14) of females that did not oviposit and in 14.3% (2 of 14) of female ticks that laid eggs, but not detected in egg clutches (100 eggs/female). DNA was also detected in tick feces collected on days 2 through 6 post-capillary tube feeding, however, dogs (n=3) did not become bacteremic or seroconvert when inoculated with tick fecal material. Therefore, trans-ovarial transmission of B. v. berkhoffii by R. sanguineus is unlikely, but further studies are needed to determine if tick fecal material can serve as a source of infection to canines.

Groups of Swiss Webster outbred mice were each inoculated with one of four bartonella strains originally isolated from Rattus spp. at doses ranging from 10(1) to 10(7) bacteria per mouse. One strain, Rn1691yn (Bartonella coopersplainensis-like), infected mice and produced bacteremias at levels up to 10(5) bacteria/ml of blood and from 3 to 8 weeks duration. A dose dependent response was also observed with differing proportions of mice bacteremic following inoculation at different doses. In addition weeks-to-months long lags in bacteremia manifestation occurred following lower dose exposures. The possibility of bacterial transmission from bacteremic mice to uninfected cagemates was assessed and no naive mice became infected from contacts with infected mice. Finally, a subset of bacteremic mice inoculated with high doses of Rn1691yn were examined histopathologically and multifocal, granulomatous lesions were detected in both liver and kidneys. The host specificity and infectivity of the strains is discussed in relation to their potential for zoonotic transmission to incidental hosts.

Botulism is a rare, life-threatening paralytic disease of both humans and animals that is caused by botulinum neurotoxins (BoNT). Botulism is confirmed in the laboratory by the detection of BoNT in clinical specimens, contaminated foods, and cultures. Despite efforts to develop an in vitro method for botulinum toxin detection, the mouse bioassay remains the standard test for laboratory confirmation of this disease. In this study, we evaluated the use of a nonlethal mouse toe-spread reflex model to detect BoNT spiked into buffer, serum, and milk samples. Samples spiked with toxin serotype A and nontoxin control samples were injected into the left and right extensor digitorum longus muscles, respectively. Digital photographs at 0,8, and 24 h were used to obtain objective measurements through effective paralysis scores, which were determined by comparing the width-to-length ratio between right and left feet. Both objective measurements and clinical observation could accurately identify over 80% of animals injected with 1 LD(50) (4.3 pg) BoNT type A within 24 h. Half of animals injected with 0.5 LD(50) BoNT type A and none injected with 0.25 LD(50) demonstrated localized paralysis. Preincubating the toxin with antitoxin prevented the development of positive effective paralysis scores, demonstrating that (1) the effect was specific for BoNT and (2) identification of toxin serotype could be achieved by using this method. These results suggest that the mouse toe-spread reflex model may be a more humane alternative to the current mouse bioassay for laboratory investigations of botulism.