The response of adipose tissues to Mycoplasma pulmonis and Sendai virus infection in C57BL/6 and DBA/2 mice.

Adipose tissues in mammals are categorized into white and brown adipose tissues in which cellular morphology, cell functions, and tissue distribution are different. White adipose tissue (WAT) plays a major role in energy reservation, while brown adipose tissue (BAT) mainly relates to the thermoregulation of the body. One interesting function of adipose tissue is the response to the infection, especially the pathogens that cause pneumonia. We have previously reported that DBA/2 (D2) mice are susceptible to pathogens causing pneumonia, Mycoplasma (M.) pulmonis and Sendai virus (SeV), whereas C57BL/6 (B6) mice are resistant to them. Furthermore, morphological alteration of mediastinal fat tissue (MFT) was seen after infection of M. pulmonis in D2 mice but not in B6 mice. In this study, we aimed to exhibit the difference in adipose tissue response in other areas, including interscapular brown adipose tissue (iBAT), inguinal white adipose tissue (ingWAT), and perigonadal WAT (perigoWAT) between resistant strain, B6 and susceptible strain, D2 after challenging them with M. pulmonis and SeV. Compared with B6 mice, D2 mice showed an increase in fat-associated lymphoid cluster in MFT, an increase in BAT in both iBAT and ingWAT after M. pulmonis and SeV infection. The results of this study indicate that pneumonia caused by M. pulmonis and SeV infection induces browning of adipocyte, suggesting that BAT plays a role in pathogen infection and inflammation.

Welfare Impact of Carbon Dioxide Euthanasia on Laboratory Mice and Rats: A Systematic Review.

Background: There has been increased concern about the suitability of CO2 as a method for euthanasia of laboratory mice and rats, including the potential discomfort, pain or distress that animals may experience prior to loss of consciousness; time to loss of consciousness; best methods for use of CO2; and the availability of better alternatives. These discussions have been useful in providing new information, but have resulted in significant confusion regarding the acceptability of CO2 for rodent euthanasia. In some cases, researchers and veterinarians have become uncertain as to which techniques to recommend or use for euthanasia of laboratory mice and rats. Methods: The International Association of Colleges of Laboratory Animal Medicine (IACLAM) convened a taskforce to examine the evidence for adverse welfare indicators in laboratory rats and mice undergoing CO2 euthanasia using a SYRCLE-registered systematic review protocol. Of 3,772 papers identified through a database search (PubMed, Web of Science, CAB Direct, Agricola, and grey literature) from 1900 to 2017, 37 studies were identified for detailed review (some including more than one species or age group), including 15 in adult mice, 21 in adult rats, and 5 in neonates of both species. Experiments or reports were excluded if they only assessed parameters other than those directly affecting animal welfare during CO2 induction and/or euthanasia. Results: Study design and outcome measures were highly variable and there was an unclear to high risk of bias in many of the published studies. Changes in the outcome measures evaluated were inconsistent or poorly differentiated. It is likely that repeated exposures to carbon dioxide inhalation are aversive to adult rats and mice, based on avoidance behavior studies; however, this effect is largely indistinguishable from aversion induced by repeated exposures to other inhalant anesthetic gasses. Conclusion: There is insufficient evidence to permit an unbiased assessment of the effect of CO2 inhalation during euthanasia on welfare indicators in laboratory mice and rats. Additional well-designed, unbiased, and adequately powered studies are needed to accurately assess the welfare of laboratory mice and rats undergoing euthanasia via CO2 gas.

Null mutation of the endothelin receptor type B gene causes embryonic death in the GK rat.

The Hirschsprung disease (HSCR) is an inherited disease that is controlled by multiple genes and has a complicated genetic mechanism. HSCR patients suffer from various extents of constipation due to dysplasia of the enteric nervous system (ENS), which can be so severe as to cause complete intestinal obstruction. Many genes have been identified as playing causative roles in ENS dysplasia and HSCR, among them the endothelin receptor type B gene (Ednrb) has been identified to play an important role. Mutation of Ednrb causes a series of symptoms that include deafness, pigmentary abnormalities, and aganglionosis. In our previous studies of three rat models carrying the same spotting lethal (sl) mutation on Ednrb, the haplotype of a region on chromosome (Chr) 2 was found to be responsible for the differing severities of the HSCR-like symptoms. To confirm that the haplotype of the responsible region on Chr 2 modifies the severity of aganglionosis caused by Ednrb mutation and to recreate a rat model with severe symptoms, we selected the GK inbred strain, whose haplotype in the responsible region on Chr 2 resembles that of the rat strain in which severe symptoms accompany the Ednrbsl mutation. An Ednrb mutation was introduced into the GK rat by crossing with F344-Ednrbsl and by genome editing. The null mutation of Ednrb was found to cause embryonic death in F2 progeny possessing the GK haplotype in the responsible region on Chr 2. The results of this study are unexpected, and they provide new clues and animal models that promise to contribute to studies on the genetic regulatory network in the development of ENS and on embryogenesis.

Profiling of cellular immune responses to Mycoplasma pulmonis infection in C57BL/6 and DBA/2 mice.

Mycoplasma infections cause respiratory tract damages and atypical pneumonia, resulting in serious problems in humans and animals worldwide. It is well known that laboratory inbred mouse strains show various susceptibility to Mycoplasma pulmonis (M. pulmonis) infection, which causes murine respiratory mycoplasmosis. In this study, we aimed to demonstrate the difference in cellular immune responses between resistant strain, C57BL/6NCrSlc (B6) and susceptible strain, DBA/2CrSlc (D2) after challenging M. pulmonis infection. D2 mice showed higher amount of bacterial proliferation in lung, higher pulmonary infiltration of immune cells such as neutrophils, macrophages, and lymphocytes, and higher levels of interleukin (IL)-1ß, IL-6, IL-17A, and tumor necrosis factor-α in bronchoalveolar lavage fluid than did B6 mice. The results of this study suggest that D2 mice are more susceptible than B6 mice to M. pulmonis infection due to a hyper-immune inflammatory response.

Analysis for genetic loci controlling protoscolex development in the Echinococcus multilocularis infection using congenic mice.

The resistance/susceptibility to Echinococcus multilocularis infection in mice is genetically controlled. However, genetic factors responsible for these differences remain unknown. Our previous study in genetic linkage analysis has revealed that there is a significant quantitative trait locus (QTL) for the establishment of cyst (Emcys1), and a highly significant QTL for the development of protoscolex of E. multilocularis larvae (Empsc1), on mouse chromosomes 6 and 1, respectively. The current study aimed to confirm these QTLs and narrow down the critical genetic region that controls resistance/susceptibility to E. multilocularis infection by establishing congenic and subcongenic lines from C57BL/6 (B6) and DBA/2 (D2) mice. For protoscolex development phenotype, two congenic lines, B6.D2-Empsc1 and D2.B6-Empsc1 were developed, where responsible QTL, Empsc1 was introgressed from D2 into B6 background and vice versa. For cyst establishment phenotype, two congenic lines, B6.D2-Emcys1 and D2.B6-Emcys1 were developed, where responsible QTL, Emcys1 was introgressed from D2 into B6 background and vice versa. Because there was no significant difference in cyst establishment between B6.D2-Emcys1 and D2.B6-Emcys1 mice after challenge with E. multilocularis, it is suggested that the Emcys1 does not solely control the cyst establishment in mouse liver. However, infection experiments with B6.D2-Empsc1 and D2.B6-Empsc1 mice showed a significant difference in protoscolex development in the cyst. It confirms that the Empsc1 controls phenotype of the protoscolex development in the cyst. Subsequently, two subcongenic lines, B6.D2-Empsc1.1 and B6.D2-Empsc1.2 from B6.D2-Emcys1 and one subcongenic line, D2.B6-Empsc1.1 from D2.B6-Empsc1 were developed to narrow down the critical region responsible for protoscolex development. From the results of infection experiments with E. multilocularis in these subcongenic mice, it is concluded that a gene responsible for protoscolex development is located between D1Mit290 (68.1 cM) and D1Mit511 (97.3 cM).

Functional analysis of duck, goose, and ostrich 2'-5'-oligoadenylate synthetase.

Up-to-date the flavivirus infection in avian taxa is not clearly defined. Several reports have demonstrated that many viruses belonging to Flaviviridae may cause diseases in poultry species; however, the susceptibility of other avian species is variable and still unclear. In human and mice, the 2'-5'-oligoadenylate synthetase (OAS) proteins are associated with resistance to the flavivirus infection as well as other virus infections. However, the avian OAS proteins are rarely studied. In our previous studies, we confirmed that the chicken OAS-like protein (chOASL) expressed OAS-enzymatic activity (the classical OAS/RNase L-dependent pathway) as well as the anti-flavivirus activity (the putative OAS/RNase L-independent pathway). Therefore, the current study aimed at functional analysis of avian OAS proteins from duck, goose, and ostrich. The duOASL, goOASL, and osOAS1 proteins expressed enzymatic activity as well as chOASL, whereas osOASL expressed little enzymatic activity. On the other hand, duOASL, goOASL, and osOASL possessed significant antiviral activity against West Nile virus (WNV)-replicon replication as well as chOASL, whereas osOAS1 did not. In addition, similar to chOASL, their antiviral activity was independent of RNase L activation. These results suggest that OASL is the only OAS protein in the duck and goose as well as chicken and possesses both OAS-enzymatic and anti-flavivirus activities, whereas the ostrich possesses both OAS1 and OASL proteins with sharing the functional activities, OAS-enzymatic and anti-flavivirus activities, respectively. It is of interest that the ostrich undergoes differential process in OAS gene evolution from other poultries and thus possesses different molecular mechanism in antiviral activity.

Verification of genetic loci responsible for the resistance/susceptibility to the Sendai virus infection using congenic mice.

Sendai virus (SeV) is one of the most important pathogens in the specific-pathogen free rodents. It is known that there are some inbred mouse strains susceptible or resistant to SeV infection. The C57BL/6 (B6) and DBA/2 (D2) mice are representative of the resistant and susceptible strains, respectively. Previous study with the quantitative trait locus (QTL) analysis identified three QTLs responsible for resistance or susceptibility to SeV infection on different chromosomes and indicated that resistance or susceptibility to SeV infection was almost predicted by genotypes of these three QTLs. In this paper, to verify the above hypothesis, congenic lines were generated as follows; B6-congenic lines carrying one of the D2 alleles of three QTLs and combination of these three QTLs, and D2-congenic lines carrying single or combination of B6 alleles of three QTLs. All these congenic lines were then challenged with SeV infection. D2 congenic lines introgressed single or combination of B6 alleles of QTLs changed to resistance to SeV infection. Especially, a D2 triple-congenic line became resistant as similar level to B6-parental strain. However, B6-congenic lines introgressed single or combination of D2 alleles of QTLs all remained to be resistant to SeV infection. Both IL-6 and TNF-α in broncho-alveolar lavage fluid of D2 triple-congenic line were decreased to the similar level of B6 mice, suggesting that this is a part of factors that D2 triple-congenic line became resistant to the similar level of B6 mice. Data obtained from these congenic mice verified that three QTLs identified previously were indeed responsible for the resistance/susceptibility to SeV infection in B6 and D2 mice.

Analysis of the Relationship Between Enzymatic and Antiviral Activities of the Chicken Oligoadenylate Synthetase-Like.

The oligoadenylate synthetase (OAS) is well known as an antiviral factor against the flavivirus infection in mammals. It is known that the oligoadenylate synthetase-like (ChOAS-L) gene is only present in the chicken genome. It has been shown in the previous report that the ChOAS-L possesses enzymatic activity to convert ATP into 2'-5'-linked oligoadenylates and antiviral activity against West Nile virus (WNV) replicon. Therefore, this study aimed to investigate the relationship between enzymatic and antiviral activities of ChOAS-L. Eight mutated ChOAS-L proteins were generated using either the site-directed mutagenesis or standard polymerase chain reaction protocol. The wild-type and mutated proteins were ectopically expressed in 293FT cells to analyze the enzymatic activity and in BHK-21 and BALB/3T3 cells to analyze the antiviral activity using WNV replicon. The results revealed that all mutated proteins showed no enzymatic activity except for ChOAS-L-AΔUbL2. However, all mutated proteins showed antiviral activity to inhibit the replication of the WNV replicon except for ChOAS-L-AΔUbL1/UbL2, which showed a partial inhibition compared to the wild-type ChOAS-L-A or other mutated proteins. These results suggest that the ChOAS-L expresses the antiflavivirus activity in a manner independent of enzymatic activity. Our results propose reconsideration of the mechanism of antiviral activity against the flavivirus replication of ChOAS-L.

The role of mouse 2',5'-oligoadenylate synthetase 1 paralogs.

The interferon-induced oligoadenylate synthetase (OAS) family is one of the most important immune response proteins to the viral infection. The OAS protein binds dsRNA and is activated to produce 2',5'-oligoadenylates, which lead to the activation of latent form of RNase L, resulting in degradation of cellular and viral RNA and inhibition of viral replication. In mice, the Oas gene family locates on chromosome 5. The mouse Oas gene locus undergoes a recent series of duplication event, leading to the presence of eight paralogs of Oas1 genes (Oas1a through Oas1h) that forms Oas gene cluster with the Oas2, Oas3 and two OasL (OasL1 and OasL2) genes. Previous studies demonstrated that the mouse Oas1b gene conferred resistance to the flavivirus infection in mice; however, the antiviral activity of other mouse Oas1 gene family is still unknown. Therefore, in the present study, we have evaluated the mouse Oas1 paralogs regarding the enzymatic activity and antiviral activity against the two neurotropic flaviviruses, West Nile virus and tick-borne encephalitis virus. The mouse Oas1 genes were cloned from C57BL/6J (B6) as well as the Oas1b derived from feral mouse strain, MSM. The obtained results demonstrated that only Oas1a and Oas1g showed enzymatic activity. Although MSM-derived Oas1b showed antiviral activity to both viruses, all B6-derived OAS paralogs did not show antiviral activity. These results suggest that Oas1a and Oas1g play a role in potentiating viral RNA-induced interferon response in the cell, whereas the Oas1b works as a specific anti-flavivirus factor unless it is mutated. However, the role of other paralogs is unknown and should wait for further investigation.

Mouse chromosome 2 harbors genetic determinants of resistance to podocyte injury and renal tubulointerstitial fibrosis.

BACKGROUND: Tensin2 deficiency results in alterations in podocytes and subsequent glomerular and tubulointerstitial injuries. However, this pathology is critically dependent on genetic background. While the Tensin2-deficient podocytes of resistant murine strains, including C57BL/6J mice, remain almost intact, susceptible murine strains with Tensin2 deficency, including ICGN mice, develop chronic kidney disease following alterations in the podocyte foot processes. In a previous study, genome-wide linkage analysis was utilized to identify the quantitative trait loci associated with the disease phenotypes on mouse chromosome 2. This study investigated the disease phenotypes of chromosome 2 consomic and subcongenic strains. RESULTS: ICGN consomic mice introgressed with chromosome 2 from the C57BL/6J mouse were generated and found to exhibit milder renal failure than that in ICGN mice. We developed 6 subcongenic strains that carry C57BL/6J-derived chromosomal segments from the consomic strain. One showed significantly milder albuminuria, another showed significantly milder tubulointerstitial injury, and the both showed significantly milder glomerular injury. CONCLUSIONS: These data indicate that mouse chromosome 2 harbors two major genes associated with the severities of nephropathy induced by Tensin2 deficiency. The proximal region on chromosome 2 contributes to the resistance to tubulointerstitial fibrosis. In contrast, the distal region on chromosome 2 contributes to the resistance to podocyte injury. This study would be helpful to discover the biological mechanism underlying the renal injury, and may lead to the identification of therapeutic targets.

QTL analysis of modifiers for pigmentary disorder in rats carrying Ednrb sl mutations.

Pigmentary variation in animals has been studied because of its application in genetics, evolution, and developmental biology. The large number of known color loci provides rich resource to elucidate the functional pigmentary system. Nonetheless, more color loci remain to be identified. In our previous study, we revealed that two different strains, namely, AGH rats and LEH rats, but which had the same null mutation of the Ednrb gene (Ednrb(sl)) showed markedly different pigmented coat ratio. This result strongly suggested that the severity of pigment abnormality was modified by genetic factor(s) in each strain. To elucidate the modifier locus of pigment disorder, we carried out whole-genome scanning for quantitative trait loci (QTLs) on 149 F2 (AGH-Ednrb(sl) × LEH-Ednrb(sl)) rats. A highly significant QTL, constituting 26% of the total pigmentation phenotype variance, was identified in a region around D7Got23 on chromosome (Chr) 7. In addition, investigation on epistatic interaction revealed significant interactions between D7Got23 and D3Rat78 and between D7Got23 and D14Mit4. Results suggested that a modified locus on Chr 7 was mainly responsible for the variance of pigmentary disorder between AGH-Ednrb(sl) rats and LEH-Ednrb(sl) rats, and two modifier loci showing epistatic interaction may, in part, influence pigment phenotype.

Does the routine handling affect the phenotype of disease model mice?

The three different mouse handling methods, picking up by tails, tunnels, and open hands were performed using the ICGN glomerulonephritis mouse and the severity of symptoms was evaluated. The handling groups exhibited a tendency of more severe symptoms than the non-handling control group. Female mice handled by their tails showed significantly more severe symptoms than the control group. In addition, we subjected the normal laboratory mice, C57BL/6 and BALB/c mice to tail and tunnel handling to assess the stress conditions. The plasma corticosterone level in the tail-handled mice was higher than that in control mice. These results indicate that handling causes stress and may affect the phenotype of disease model mice.

Laboratory Animal Laws, Regulations, Guidelines and Standards in China Mainland, Japan, and Korea.

China, Japan, and Korea have spent decades developing and amending laws, regulations, and guidelines to address the humane care and use of laboratory animals. This process began in 1983 in China, 1973 in Japan, and 1991 in Korea and has continued to the present. The governmental oversight of research varies between these countries, ranging from regulations by multiple levels of government in China to self-regulation under multiple government guidelines in Japan. Common to all is incorporation of the internationally recognized principles of the 3Rs: replacement, reduction and refinement. This paper reviews how the framework of laws, regulations, and guidelines evolved in each of these countries, their current status, and the expectation that they will continue to evolve.

Genetic variation in the GDNF promoter affects its expression and modifies the severity of Hirschsprung's disease (HSCR) in rats carrying Ednrb(sl) mutations.

Glial cell line-derived neurotrophic factor (GDNF) is necessary for the migration of neural crest stem cells in the gut. However, mutations in GDNF per se are deemed neither necessary nor sufficient to cause Hirschsprung's disease (HSCR). In a previous study, a modifier locus on chromosome 2 in rats carrying Ednrb(sl) mutations was identified, and several mutations in the putative regulatory region of the Gdnf gene in AGH-Ednrb(sl) rats were detected. Specifically, the mutation -232C>T has been shown to be strongly associated with the severity of HSCR. In the present study, the influence of genetic variations on the transcription of the Gdnf gene was tested using dual-luciferase assay. Results showed that the mutation -613C>T, located near the mutation -232C>T in AGH-Ednrb(sl) rats, decreased Gdnf transcription in an in vitro dual-luciferase expression assay. These data suggested an important role of -613C in Gdnf transcription. Expression levels of the Gdnf gene may modify the severity of HSCR in rats carrying Ednrb(sl) mutations.

Genetic background-dependent diversity in renal failure caused by the tensin2 gene deficiency in the mouse.

Tensin2 (Tns2) is thought to be a component of the cytoskeletal structures linking actin filaments with focal adhesions and is known to play a role as an intracellular signal transduction mediator through integrin in podocytes, although the mechanism by which it functions remains unclear. A Tns2-null mutation (nph) leads to massive albuminuria following podocyte foot process effacement in the ICGN mice, the origin of the mutation, and the DBA/2J (D2) mice, but not in the C57BL/6J (B6) mice or 129(+Ter)/SvJcl (129T) mice. Elucidating the reasons for these differences in diverse genetic backgrounds could help in unraveling Tns2 function in podocytes. We produced congenic mice in which Tns2(nph) was introgressed into a FVB/NJ background (FVB-Tns2(nph)), and evaluated the progression of kidney disease. FVB-Tns2(nph) mice developed albuminuria, renal fibrosis and renal anemia as seen in ICGN mice. The FVB-Tns2(nph) mice demonstrated podocyte foot process alteration under an electron microscope by as early as 4 weeks of age. This revealed that FVB strain is susceptible to Tns2-deficiency.

Identification of multiple genetic loci in the mouse controlling immobility time in the tail suspension and forced swimming tests.

Depression is one of the most famous psychiatric disorders in humans in all over the countries and considered a complex neurobehavioral trait and difficult to identify causal genes. Tail suspension test (TST) and forced swimming test (FST) are widely used for assessing depression-like behavior and antidepressant activity in mice. A variety of antidepressant agents are known to reduce immobility time in both TST and FST. To identify genetic determinants of immobility duration in both tests, we analyzed 101 F2 mice from an intercross between C57BL/6 and DBA/2 strains. Quantitative trait locus (QTL) mapping using 106 microsatellite markers revealed three loci (two significant and one suggestive) and five suggestive loci controlling immobility time in the TST and FST, respectively. Results of QTL analysis suggest a broad description of the genetic architecture underlying depression, providing underpinnings for identifying novel molecular targets for antidepressants to clear the complex genetic mechanisms of depressive disorders.

Identification of genetic loci affecting the severity of symptoms of Hirschsprung disease in rats carrying Ednrbsl mutations by quantitative trait locus analysis.

Hirschsprung's disease (HSCR) is a congenital disease in neonates characterized by the absence of the enteric ganglia in a variable length of the distal colon. This disease results from multiple genetic interactions that modulate the ability of enteric neural crest cells to populate developing gut. We previously reported that three rat strains with different backgrounds (susceptible AGH-Ednrbsl/sl, resistant F344-Ednrbsl/sl, and LEH-Ednrbsl/sl) but the same null mutation of Ednrb show varying severity degrees of aganglionosis. This finding suggests that strain-specific genetic factors affect the severity of HSCR. Consistent with this finding, a quantitative trait locus (QTL) for the severity of HSCR on chromosome (Chr) 2 was identified using an F2 intercross between AGH and F344 strains. In the present study, we performed QTL analysis using an F2 intercross between the susceptible AGH and resistant LEH strains to identify the modifier/resistant loci for HSCR in Ednrb-deficient rats. A significant locus affecting the severity of HSCR was also detected within the Chr 2 region. These findings strongly suggest that a modifier gene of aganglionosis exists on Chr 2. In addition, two potentially causative SNPs (or mutations) were detected upstream of a known HSCR susceptibility gene, Gdnf. These SNPs were possibly responsible for the varied length of gut affected by aganglionosis.

Quantitative trait Loci for resistance to the congenital nephropathy in tensin 2-deficient mice.

The ICR-derived glomerulonephritis (ICGN) mouse is a chronic kidney disease (CKD) model that is characterized histologically by glomerulosclerosis, vascular sclerosis and tubulointerstitial fibrosis, and clinically by proteinuria and anemia, which are common symptoms and pathological changes associated with a variety of kidney diseases. Previously, we performed a quantitative trait locus (QTL) analysis to identify the causative genes for proteinuria in ICGN mice, and found a deletion mutation of the tensin 2 gene (Tns2nph, MGI no: 2447990). Interestingly, the congenic strain carrying the Tns2nph mutation on a C57BL/6J (B6) genetic background exhibited milder phenotypes than did ICGN mice, indicating the presence of several modifier genes controlling the disease phenotype. In this study, to identify the modifier/resistant loci for CKD progression in Tns2-deficient mice, we performed QTL analysis using backcross progenies from susceptible ICGN and resistant B6 mice. We identified a significant locus on chromosome (Chr) 2 (LOD = 5.36; 31 cM) and two suggestive loci on Chrs 10 (LOD = 2.27; 64 cM) and X (LOD = 2.65; 67 cM) with linkage to the severity of tubulointerstitial injury. One significant locus on Chr 13 (LOD = 3.49; approximately 14 cM) and one suggestive locus on Chr 2 (LOD = 2.41; 51 cM) were identified as QTLs for the severity of glomerulosclerosis. Suggestive locus in BUN was also detected in the same Chr 2 region (LOD = 2.34; 51 cM). A locus on Chr 2 (36 cM) was significantly linked with HGB (LOD = 4.47) and HCT (LOD = 3.58). Four novel epistatic loci controlling either HGB or tubulointerstitial injury were discovered. Further genetic analysis should lead to identification of CKD modifier gene(s), aiding early diagnosis and providing novel approaches to the discovery of drugs for the treatment and possible prevention of kidney disease.

Development of an ELISA using a recombinant P46-like lipoprotein for diagnosis of Mycoplasma pulmonis infection in rodents.

Mycoplasma pulmonis is one of the most prevalent bacterial pathogens that infects laboratory mice and rats. To develop an M. pulmonis-specific antigen for serological diagnosis, we cloned the cDNA of P46-like lipoprotein (P46L), an M. pulmonis putative periplasmic protein. P46L is a homolog of P46, an M. hyopneumoniae antigen. We produced recombinant P46L fused to glutathione S-transferase (GST) in Escherichia coli. Immunoblot analysis revealed that sera from Mycoplasma-infected mice and rats contained anti-P46L antibodies. We developed an ELISA using the recombinant P46L-GST protein as an antigen. Thirteen of the 14 samples from rats naturally infected with M. pulmonis were determined to be positive according to the commercial ELISA (MONILISA Myco) and positive by our ELISA. Furthermore, 18/19 samples from mice experimentally infected with M. pulmonis were positive using our P46L-GST ELISA. In contrast, only 8/19 samples from infected mice were positive by the commercial ELISA. Our results indicate that P46L-GST was an appropriate antigen for developing a serological test to determine M. pulmonis infection in laboratory mice and rats.

Epitope mapping of the nucleocapsid protein of sendai virus and application of antigenic epitopes for the ELISA-based diagnosis of sendai virus infection.

Sendai virus (SeV) is one of the most prevalent viral pathogens infecting laboratory mice and rats. To date, mature SeV virions have been used as antigens for serological diagnosis. To develop antigens that are more specific and easier to prepare for diagnosis, we examined the antigenic sites in the nucleocapsid protein (NP) of SeV with antisera from experimentally SeV-infected mice and a peptide array membrane containing overlapping 10-mer peptides covering the entire NP. We found antigenic linear sequences in two regions, amino acids 120-160 and 420-500, of the SeV-NP. From these antigenic sequences, we applied two synthesized peptides, IVKTRDMEYERTTEWL and FVTLHGAERLEEETNDE, which correspond to positions 119-134 and 458-474 of the SeV-NP, respectively, as antigens in an enzyme-linked immunosorbent assay (ELISA). Evaluation of the ELISAs using these peptides revealed that they were specific to anti-SeV antisera. Furthermore, the ELISAs using these peptides were able to distinguish between SeV-positive and SeV-negative mouse sera to the same extent as a commercial ELISA kit. These results indicate that these peptides are useful for the serological diagnosis of SeV infection.