Specific Pathogen-free Animals Research Articles

Impact of microbial diversity on inflammatory cytokines and respiratory pattern measured in whole-body plethysmography in guinea pig models.

This study investigates the breathing patterns and immune status of guinea pigs raised under specific pathogen-free (SPF) conditions compared to conventionally bred (CON). Breathing pattern parameters were assessed using whole-body plethysmography (WBP) during quiet breathing and saline nebulisation. Blood and bronchoalveolar lavage fluid (BALF) were analysed for white blood cell, neutrophil and eosinophil counts, and cytokine levels (TNF-α, IL-1β, IL-4). SPF guinea pigs exhibited higher tidal volume, expired volume, minute volume, and airflow parameters than CON guinea pigs. The immune analysis revealed lower white blood cell counts and IL-4 levels in SPF guinea pigs. These findings indicate that SPF guinea pigs have different respiratory and immune responses than CON guinea pigs. The study highlights that the maturation processes affecting breathing pattern parameters in SPF guinea pigs differ significantly from those in CON guinea pigs. This suggests potential limitations of SPF animals in respiratory physiology research due to their different immune and respiratory responses.

Moving from guideline recommendations to practical, reasonable proposals: Biosafety considerations, breeding performance and challenges during specific pathogen-free animal facility enlargement.

Extending an existing animal facility is a challenging process that requires consideration of both engineering and biological aspects. In this sense, integration with ongoing activities must not alter the animals' microbiological condition or welfare, as they usually remain in the facility while these activities occur. The objective of this work was to describe and evaluate the practical biosafety considerations during the enlargement of a specific pathogen-free (SPF) rodent facility. Our facility breeds rats and mice free of a list of zoonotic and common rodent pathogens, comprising 6 ectoparasites, 13 endoparasites, 25 bacteria and 23 viruses. In this project, the new SPF area was connected to an old but still working SPF rodent facility through the original clean corridor. The old clean corridor remained sealed throughout the project, and it was not opened until the new area was finished and fully equipped, all the new rooms were cleaned and disinfected, and the environment was evaluated for the presence of pathogens. Timing during the project was essential, as avoidance of the period of high animal production and demand was sought. The microbiological controls showed no growth of microorganisms in any new room. Thus, the applied procedures were deemed effective. It was concluded that protocols should be carefully planned in order to maintain the SPF condition and animal welfare.

Investigation of natural infection of BALB C mice by Bartonella henselae

Specific Pathogen-Free (SPF) animals are bred and maintained to exclude pathogens associated with significant morbidity or mortality, which may pose a risk to research replicability. The BALB/c strain is distributed globally and is among the most commonly used inbred strains in immunology and infectious disease research. Despite being a widely distributed bacterium that causes chronic infection, Bartonella henselae infection has not been investigated in any protocol that characterizes SPF animals. The objective of this study was to investigate the potential natural infection of laboratory animals of the BALB/c lineage by B. henselae. To achieve this, ten immunocompetent BALB/c mice were obtained directly from the bioterium and euthanized for collection of samples, including blood, skin, spleen, liver, heart, eye, kidney, intestine, esophagus, and brain. DNA was extracted using a commercial kit and tested via nested PCR for the ftsZ gene, as well as conventional PCR and qualitative real-time PCR using Sybr® Green for the citrate synthase gene (gltA), all specific reactions for B. henselae. All animals showed detection of B. henselae DNA in at least two different reactions in different tissues. The sequenced amplicons showed 100% similarity to B. henselae. The use of mice infected by B. henselae in experiments is undesirable, as the bacteria can affect several aspects of the animal's physiology and consequently influence the results of the project, especially when subjected to immunosuppression. More studies are needed to understand and confirm the natural infection in experimental animals by Bartonella spp. To date, no additional published reports of contamination of experimental animals by these bacteria have been identified.

α-synuclein overexpression and the microbiome shape the gut and brain metabolome in mice

Pathological forms of α-synuclein contribute to synucleinopathies, including Parkinson’s disease (PD). Most cases of PD arise from gene-environment interactions. Microbiome composition is altered in PD, and gut bacteria are causal to symptoms in animal models. We quantitatively profiled nearly 630 metabolites in the gut, plasma, and brain of α-synuclein-overexpressing (ASO) mice, compared to wild-type (WT) animals, and comparing germ-free (GF) to specific pathogen-free (SPF) animals (n = 5 WT-SPF; n = 6 ASO-SPF; n = 6 WT-GF; n = 6 ASO-GF). Many differentially expressed metabolites in ASO mice are also dysregulated in human PD patients, including amine oxides, bile acids and indoles. The microbial metabolite trimethylamine N-oxide (TMAO) strongly correlates from the gut to the plasma to the brain in mice, notable since TMAO is elevated in the blood and cerebrospinal fluid of PD patients. These findings uncover broad metabolomic changes that are influenced by the intersection of host genetics and microbiome in a mouse model of PD.

Cutting Edge: First Lung Infection Permanently Enlarges Lymph Nodes and Enhances New T Cell Responses.

Humans experience frequent respiratory infections. Immunology and vaccinology studies in mice are typically performed in naive specific pathogen-free animals responding to their very first respiratory challenge. We found that the first respiratory infection induces lifelong enlargement of the lung-draining mediastinal lymph nodes (medLNs). Furthermore, infection-experienced medLNs supported better naive T cell surveillance and effector responses to new unrelated infections that exhibited more biased accumulation and memory establishment within the lung. Moreover, we observed that weight loss induced by influenza infection was substantially reduced in mice that had recovered from a previous unrelated respiratory viral challenge. These data show that the lack of infectious history and corresponding medLN hypoplasia in specific pathogen-free mice alter their immune response to lung infections. Preclinical vaccination and immunology studies should consider the previous infectious experience of the model organism.

The effect of oral butyrate on colonic short-chain fatty acid transporters and receptors depends on microbial status.

Butyrate, a metabolite produced by gut bacteria, has demonstrated beneficial effects in the colon and has been used to treat inflammatory bowel diseases. However, the mechanism by which butyrate operates remains incompletely understood. Given that oral butyrate can exert either a direct impact on the gut mucosa or an indirect influence through its interaction with the gut microbiome, this study aimed to investigate three key aspects: (1) whether oral intake of butyrate modulates the expression of genes encoding short-chain fatty acid (SCFA) transporters (Slc16a1, Slc16a3, Slc16a4, Slc5a8, Abcg2) and receptors (Hcar2, Ffar2, Ffar3, Olfr78, Olfr558) in the colon, (2) the potential involvement of gut microbiota in this modulation, and (3) the impact of oral butyrate on the expression of colonic SCFA transporters and receptors during colonic inflammation. Specific pathogen-free (SPF) and germ-free (GF) mice with or without DSS-induced inflammation were provided with either water or a 0.5% sodium butyrate solution. The findings revealed that butyrate decreased the expression of Slc16a1, Slc5a8, and Hcar2 in SPF but not in GF mice, while it increased the expression of Slc16a3 in GF and the efflux pump Abcg2 in both GF and SPF animals. Moreover, the presence of microbiota was associated with the upregulation of Hcar2, Ffar2, and Ffar3 expression and the downregulation of Slc16a3. Interestingly, the challenge with DSS did not alter the expression of SCFA transporters, regardless of the presence or absence of microbiota, and the effect of butyrate on the transporter expression in SPF mice remained unaffected by DSS. The expression of SCFA receptors was only partially affected by DSS. Our results indicate that (1) consuming a relatively low concentration of butyrate can influence the expression of colonic SCFA transporters and receptors, with their expression being modulated by the gut microbiota, (2) the effect of butyrate does not appear to result from direct substrate-induced regulation but rather reflects an indirect effect associated with the gut microbiome, and (3) acute colon inflammation does not lead to significant changes in the transcriptional regulation of most SCFA transporters and receptors, with the effect of butyrate in the inflamed colon remaining intact.

Evaluation of the Hairballs in the Gastrointestinal Tract and Urinary Stones in Razi Institute Laboratory Rabbits

Background: Usually, the daily self-grooming by rabbits leads to fur accumulation in the animal’s stomach. Since rabbit hair is looser than other animals and constantly licks their body, the fur can be pulled out easily. On the other hand, rabbits are susceptible to urinary stone formation. Objectives: This study was designed to investigate the presence of hairballs and urinary stones in Razi Institute Laboratory rabbits. Methods: During the 1 year, the albino Dutch laboratory rabbit colony, in research, breeding, and production of the Laboratory Animals Department of Razi Institute, including 106 males, 287 females, and 166 kittens, were monitored. After the necropsy of the selected animals, the gastrointestinal tract (stomach and intestines) were examined for the presence of hair and hairballs. Then the urinary system (kidneys, ureter, urinary bladder, and urethra) was examined for any urinary stones. Results: No symptoms of anorexia, lethargy, abdominal pain, weight loss, decrease and abnormal stools were observed in them, and also no mortality occurred in the whole colony. All samples’ stomach was full, indicating enough eating. No gas or congested spots, or hemorrhage were observed in the intestines. The amount and consistency of stool in the intestines were normal. In none of the samples, hairballs were observed, but in most rabbits’ stomachs (both sexes), a small amount of hair was observed in the stomach contents. Also, no symptoms of urinary stones were observed in the colony of the studied rabbits. Conclusion: Balanced diet, supply of nutritional requirements, and the absence of any stressors in breeding environments have played a key role and prevented many diseases, such as hairballs and urinary stones. No observation of urinary stones in this study could lead to the hypothesis that infection with the bacteria that cause urinary stones in the studied rabbits was eliminated or non-pathogenic, indicating specific pathogen-free animals. However, bacterial and other infectious agent monitoring should be specialized.

FcRγ- NK Cell Induction by Specific Cytomegalovirus and Expansion by Subclinical Viral Infections in Rhesus Macaques.

"Adaptive" NK cells, characterized by FcRγ deficiency and enhanced responsiveness to Ab-bound, virus-infected cells, have been found in certain hCMV-seropositive individuals. Because humans are exposed to numerous microbes and environmental agents, specific relationships between hCMV and FcRγ-deficient NK cells (also known as g-NK cells) have been challenging to define. Here, we show that a subgroup of rhesus CMV (RhCMV)-seropositive macaques possesses FcRγ-deficient NK cells that stably persist and display a phenotype resembling human FcRγ-deficient NK cells. Moreover, these macaque NK cells resembled human FcRγ-deficient NK cells with respect to functional characteristics, including enhanced responsiveness to RhCMV-infected target in an Ab-dependent manner and hyporesponsiveness to tumor and cytokine stimulation. These cells were not detected in specific pathogen-free (SPF) macaques free of RhCMV and six other viruses; however, experimental infection of SPF animals with RhCMV strain UCD59, but not RhCMV strain 68-1 or SIV, led to induction of FcRγ-deficient NK cells. In non-SPF macaques, coinfection by RhCMV with other common viruses was associated with higher frequencies of FcRγ-deficient NK cells. These results support a causal role for specific CMV strain(s) in the induction of FcRγ-deficient NK cells and suggest that coinfection by other viruses further expands this memory-like NK cell pool.

Liver proteomic analysis reveals the key proteins involved in host immune response to sepsis.

Sepsis is a serious infection-induced response in the host, which can result in life-threatening organ dysfunction. It is of great importance to unravel the relationship between sepsis and host immune response and its mechanisms of action. Liver is one of the most vulnerable organs in sepsis, however, the specific pathogenesis of septic liver injury has not been well understood at the protein level. A total of 12 healthy Sprague-Dawley (SD) male rats aged from 6 to 8 weeks were adaptively housed in individual cages in the specific pathogen free animal room. These lab rats were grouped into two groups: treatment (N = 9) and control (N = 3) groups; only three mice from the treatment group survived and were used for subsequent experiments. A TMT-based proteomic analysis for liver tissue was performed in the septic rat model. A total of 37,012 unique peptides were identified, and then 6,166 proteins were determined, among which 5,701 were quantifiable. Compared to the healthy control group, the septic rat group exhibited 162 upregulated and 103 downregulated differentially expressed proteins (DEPs). The upregulated and downregulated DEPs were the most significantly enriched into the complement and coagulation cascades and metabolic pathways. Protein-protein interaction (PPI) analysis further revealed that the upregulated and downregulated DEPs each clustered in a PPI network. Several highly connected upregulated and downregulated DEPs were also enriched into the complement and coagulation cascades pathways and metabolic pathways, respectively. The parallel reaction monitoring (PRM) results of the selected DEPs were consistent with the results of the TMT analysis, supporting the proteomic data. Our findings highlight the roles of complement and coagulation cascades and metabolic pathways that may play vital roles in the host immune response. The DEPs may serve as clinically potential treatment targets for septic liver injury.

Rapid Detection of DNA and RNA Shrimp Viruses Using CRISPR-Based Diagnostics.

Timely detection of persistent and emerging pathogens is critical to controlling disease spread, particularly in high-density populations with increased contact between individuals and limited-to-no ability to quarantine. Standard molecular diagnostic tests for surveying pathogenic microbes have provided the sensitivity needed for early detection, but lag in time-to-result leading to delayed action. On-site diagnostics alleviate this lag, but current technologies are less sensitive and adaptable than lab-based molecular methods. Towards the development of improved on-site diagnostics, we demonstrated the adaptability of a loop-mediated isothermal amplification-CRISPR coupled technology for detecting DNA and RNA viruses that have greatly impacted shrimp populations worldwide; White Spot Syndrome Virus and Taura Syndrome Virus. Both CRISPR-based fluorescent assays we developed showed similar sensitivity and accuracy for viral detection and load quantification to real-time PCR. Additionally, both assays specifically targeted their respective virus with no false positives detected in animals infected with other common pathogens or in certified specific pathogen-free animals. IMPORTANCE The Pacific white shrimp (Penaeus vannamei) is one of the most valuable aquaculture species in the world but has suffered major economic losses from outbreaks of White Spot Syndrome Virus and Taura Syndrome Virus. Rapid detection of these viruses can improve aquaculture practices by enabling more timely action to be taken to combat disease outbreaks. Highly sensitive, specific, and robust CRISPR-based diagnostic assays such as those developed here have the potential to revolutionize disease management in agriculture and aquaculture helping to promote global food security.

Cough response in specific pathogen-free guinea pig animal model

Laboratory animal science associations recommend the usage of specific pathogen-free (SPF) animals in experiments to achieve appropriate results. However, their non-natural microbiome might represent an obstacle in the successful translation of obtained results into clinical practice, since the microbiome contributes to the regulation of the host’s physiology. Inconsistent results, even in repeated experiments in separate animal groups, irrespective of the season or sex led us to assess the cough response of SPF animal variables and compare it to wild-type animals. The dose-response curves in citric acid and capsaicin cough challenge construction shows significantly increased cough threshold in SPF animals compared to wild-type animals of both sexes. The cause of cough response alteration in SPF animals is not presently known and thus their usage in basic cough research remains questionable. The relationship between airway reflexes and the airway microbiome has never been studied hence we propose that it might be microbiome-related.

The impact of bacterial exposure in early life on lung surfactant gene expression, function and respiratory rate in germ-free mice

Early-life changes to lung and gut microbiota have been linked to alterations in immune responses that may lead to pulmonary diseases later in life. Associations between early-life microbiota, germ-free status, lung gene expression, lung development and function are not well described. In this study, we compare early-life lung gene transcription under germ-free and different perinatal microbial exposures, and analyze with a predetermined focus on lung capacity and lung surfactant. We also analyze the later-in-life physiological measures of breathing patterns and lung surfactant function between the germ-free, gnotophoric and gnotobiotic offspring. To achieve this, we kept pregnant BALB/c germ-free mice in separate germ-free isolators until exposure to either A: no exposure (GF), B: Bifidobacterium animalis ssp. Lactis (BI04) or C: full cecum content harvested from other female SPF mice (Cecum). Subsequently, perinatally exposed offspring were used for the analyses. Lung tissue transcriptomics analysis was done at postnatal day 10 (PNday10) at the first phase of lung alveolar development. Head-out plethysmography for breathing pattern analysis was performed on the siblings at PNday23 followed by lung surfactant collection. The function of the collected lung surfactant was then analyzed ex vivo using the constrained drop surfactometer. Our results show that lung transcriptomics had differentially expressed genes related to surfactant turnover between groups and sex at PNday10. They also show that the GF and BI04 animals had lower respiratory rate than Cecum mice, or compared to age-matched specific pathogen-free (SPF) reference animals. We also see changes in lung surfactant function ex vivo. The overall conclusions are that 10-day-old GF mice do not have a markedly different lung gene transcription compared to gnotophoric or gnotobiotic mice, but genes related to surfactant metabolism are among the few differentially expressed genes. We show here for the first time that early-life microbiome status correlates with early-life surfactant-gene transcription and to later-in-life lung surfactant function and associated respiratory-rate changes in mice.

Immune dysfunction following severe trauma: A systems failure from the central nervous system to mitochondria.

When a traumatic injury exceeds the body’s internal tolerances, the innate immune and inflammatory systems are rapidly activated, and if not contained early, increase morbidity and mortality. Early deaths after hospital admission are mostly from central nervous system (CNS) trauma, hemorrhage and circulatory collapse (30%), and later deaths from hyperinflammation, immunosuppression, infection, sepsis, acute respiratory distress, and multiple organ failure (20%). The molecular drivers of secondary injury include damage associated molecular patterns (DAMPs), pathogen associated molecular patterns (PAMPs) and other immune-modifying agents that activate the hypothalamic-pituitary-adrenal (HPA) axis and sympathetic stress response. Despite a number of drugs targeting specific anti-inflammatory and immune pathways showing promise in animal models, the majority have failed to translate. Reasons for failure include difficulty to replicate the heterogeneity of humans, poorly designed trials, inappropriate use of specific pathogen-free (SPF) animals, ignoring sex-specific differences, and the flawed practice of single-nodal targeting. Systems interconnectedness is a major overlooked factor. We argue that if the CNS is protected early after major trauma and control of cardiovascular function is maintained, the endothelial-glycocalyx will be protected, sufficient oxygen will be delivered, mitochondrial energetics will be maintained, inflammation will be resolved and immune dysfunction will be minimized. The current challenge is to develop new systems-based drugs that target the CNS coupling of whole-body function.

DAP10 integration in CAR-T cells enhances the killing of heterogeneous tumors by harnessing endogenous NKG2D

Although chimeric antigen receptor T (CAR-T) cells have achieved remarkable successes in hematological malignancies, the efficacies of CAR-T cells against solid tumors remains unsatisfactory. Heterogeneous antigen expression is one of the obstacles on its effective elimination of solid cancer cells. DNAX-activating protein 10 (DAP10) interacts with natural killer group 2D (NKG2D), acting as an adaptor that targets various malignant cells for surveillance. Here, we designed a DAP10 chimeric receptor that utilized native NKG2D on T cells to target NKG2D ligand-expressing cancer cells. We then tandemly incorporated it with anti-glypican 3 (GPC3) single-chain variable fragment (scFv) to construct a dual-antigen-targeting system. T cells expressing DAP10 chimeric receptor (DAP10-T cells) displayed with an enhancement on both cytotoxicity and cytokine secretion against solid cancer cell lines, and its tandem connection with anti-GPC3 scFv (CAR GPC3-DAP10-T cells) exhibited a dual-antigen-targeting capacity on eliminating heterogeneous cancer cells in vitro and suppressing the growth of heterogeneous cancer in vivo. Thus, this novel dual-targeting system enabled a high efficacy on killing cancer cells and extended the recognition profile of CAR-T cells toward tumors, which providing a potential strategy on treatment of solid cancer clinically.

Temperature and Tumor Microenvironment Dual Responsive Mesoporous Magnetic Nanospheres for Magnetothermal Effect-Induced Cancer Theranostics

Temperature and Tumor Microenvironment Dual Responsive Mesoporous Magnetic Nanospheres for Magnetothermal Effect-Induced Cancer Theranostics

Polymicrobial oral conventionalization model in mice.

The aim of this study is to describe a new polymicrobial oral conventionalization protocol in mice. Oral biofilm samples were collected from wild C57 BL/6 mice (WG), which had not been previously submitted to any experimental procedure. The contents of these samples were used for inoculation in the oral cavity of specific pathogen free (SPF) animals. This inoculation was repeated 3 times. Qualitative cytological analyses were performed in the days 0, 16 and 80 of the experimental protocol, to check the presence or absence of microorganisms, their morphology and staining characteristics on the oral cavity of the animals. At the end of this study, was observed a combination of oral bacterial microbiota of SPF animals and wild animals in the conventionalized group (CONV). Samples collected from CONV mice on day 16, a period in which these animals had been previously inoculated 3 times with wild mouse microbiota, showed a greater amount of Gram-positive cocci, as seen in SPF animals. In addition, Gram-negative cocci were present, although in a much smaller proportion than previously seen in wild mice. On the 80th experimental day, CONV animals showed a predominance of Gram-positive cocci and bacilli. Filamentous bacteria were also seen in this group. The conventionalization of SPF animals using the technique with inoculum from the resident microbiota of wild mice proved to be an effective, low-cost and easily reproducible technique. The conventionalized animals showed the colonization of a microbiota similar to wild animals up to 80days of experiment.

Intestinal Microbiota and Serum Metabolic Profile Responded to Two Nutritional Different Diets in Mice.

There is an interaction and bidirectional selection between dietary intake and gut microbiota due to the different efficiency of nutrients in the gut. The nutritional composition of germ-free (GF) diets differs significantly from specific pathogen-free (SPF) diets. There is, however, no data revealing how SPF animals from the same microbial background respond to them and if they affect the host. We examined the growth of SPF mice on the GF diet and found that it reduced body weight, intestinal length and intestinal morphology. Interestingly, the GF diet increased the level of pro-inflammatory bacteria in the gut of SPF mice, including Proteobacteria, Burkholderiaceae, Alloprevotella and Parasutterella. Furthermore, GF diets caused significant increases in malondialdehyde (MDA), IL-1β, IL-6, and D-lactate levels in the serum of SPF mice and significantly altered their serum metabolic profile, especially amino acid metabolism. In conclusion, GF diets are not suitable for the growth and development of SPF mice. These findings, based on the role of gut microbiota in diet selection, provide new insights into the scientific and rational use of experimental animal diets.

A standardized gnotobiotic mouse model harboring a minimal 15-member mouse gut microbiota recapitulates SOPF/SPF phenotypes

Mus musculus is the classic mammalian model for biomedical research. Despite global efforts to standardize breeding and experimental procedures, the undefined composition and interindividual diversity of the microbiota of laboratory mice remains a limitation. In an attempt to standardize the gut microbiome in preclinical mouse studies, here we report the development of a simplified mouse microbiota composed of 15 strains from 7 of the 20 most prevalent bacterial families representative of the fecal microbiota of C57BL/6J Specific (and Opportunistic) Pathogen-Free (SPF/SOPF) animals and the derivation of a standardized gnotobiotic mouse model called GM15. GM15 recapitulates extensively the functionalities found in the C57BL/6J SOPF microbiota metagenome, and GM15 animals are phenotypically similar to SOPF or SPF animals in two different facilities. They are also less sensitive to the deleterious effects of post-weaning malnutrition. In this work, we show that the GM15 model provides increased reproducibility and robustness of preclinical studies by limiting the confounding effect of fluctuation in microbiota composition, and offers opportunities for research focused on how the microbiota shapes host physiology in health and disease.

Nonhuman primate models for SARS-CoV-2 Research: Managing demand for specific-pathogen-free (SPF) animals.

The SARS-CoV-2/COVID-19 pandemic has significantly increased the demand for specific-pathogen-free (SPF) nonhuman primates (NHPs) for development of vaccines and therapeutics, thus straining the supply of these animals for biomedical research studies. Non-SPF animals, which are available in greater numbers and include well-characterized primate species, should be considered in lieu of limited SPF animals for appropriate research studies.

Serum Samples from Co-Infected and Domestic Cat Field Isolates Nonspecifically Bind FIV and Other Antigens in Enzyme-Linked Immunosorbent Assays.

We evaluated enzyme-linked immunosorbent assay (ELISA) specificity for measuring seroantibody responses to two types of retroviral infections in domestic cats: feline immunodeficiency virus (FIV) and feline foamy virus (FFV). We compared the seroreactivity of specific pathogen-free (SPF) cat sera, sera from SPF cats inoculated with either FIV or FFV, and field isolates (e.g., shelter or privately owned cats). Sera from SPF cats experimentally infected with the cognate virus had significantly lower background in both FIV and FFV ELISAs compared to sera from negative field isolates. ELISA values for SPF cats exposed to either FIV or FFV tended to have higher OD values on the opposite ELISA antigen plate. FIV nonspecific background absorbance was greater than that of FFV, and 10 of 15 sera samples from FIV seronegative field samples were measured in the indeterminant range. These findings highlight that exposure to off-target pathogens elicit antibodies that may nonspecifically bind to antigens used in binding assays; therefore, validation using sera from SPF animals exposed during controlled infection results in the setting of a cutoff value that may be inappropriately low when applied to field samples. Our work also suggests that infection of domestic cats with pathogens other than FIV results in antibodies that cross-react with the FIV Gag antigen.