C. T. Liang*,†, S. C. Wu*, Y. T. Huang*, Y. C. Lin*, W. J. Chang*, J. Y. Chou‡, S. C. Liang*,‡ and C. H. Liu†
*National Laboratory Animal Center, National Applied Research Laboratories, Nan-Kang, Taipei 115,†Department and Graduate Institute of Veterinary Medicine, College of Bioresources and Agriculture, National Taiwan University,
Taipei 106, and‡Laboratory Animal Center, National Defense Medical Center, Taipei, Taiwan, ROC
Summary
This study established a modified alkaline phosphatase-labelled avidin-biotin-complex (ABC-AP) method for diagnosis of mouse hepatitis virus (MHV) and Mycoplasma pulmonis infection from formalin-fixed, paraffin wax-embedded sections, murine antibody-positive serum being used as the primary reagent. With this method, MHV antigen in cAnNCrj.Cg-Foxn1nu/Foxn1numice and M. pulmonis antigen in Wistar rats were immunolabelled in tissue sections. MHV antigen was clearly detected in samples of liver, stomach, caecal and colonic mucosa, and spleen. M. pulmonis antigen was demonstrated on the luminal surface of bronchiolar epithelial cells. This method may prove useful in diagnosis when commercial antisera are unavailable or when immunosuppression prevents serological diagnosis.
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Keywords: bacterial infection; mouse; mouse hepatitis virus; Mycoplasma pulmonis; viral infection
Introduction
Avidin-biotin-complex immunohistochemistry offers a sensitive, reliable method for the detection of pathogens in tissues. Mouse hepatitis virus (MHV) and Mycoplasma pulmonis are the most prevalent pathogens of laboratory mice and rats (National Research Council, 1991). Respiratory strains of MHV infect the nasal mucosa and then spread to the liver, lymphoid tissue, uterus, placenta, peritoneum, brain, vascular endothelium and bone marrow by the lymphatic or vascular route, or directly via olfactory pathways from the nose to the brain. Enterotropic strains of MHV usually infect the intestinal mucosal epithelium
and nasal passages, with less involvement of other tissues (National Research Council, 1991;
Compton et al., 1993; Liang et al., 1995).
M. pulmonis, an extracellular pathogen of mice and rats, preferentially colonizes the luminal sur-face of respiratory epithelium, the middle ear and endometrium (National Research Council, 1991;
Percy and Barthold, 1993). Infection is usually diagnosed by microbial isolation, serological test-ing and histological examination. Microbial iso-lation requires multisite culture for reliable results, and serological methods are hampered by cross-reactivity between different species of mycoplasma (Cassell et al., 1981). Serological testing is useful during the active and convalescent phases of disease (Feldman, 2001), but screening of immu-nodeficient mice is unreliable (Casebolt et al., 1997). Immunohistochemistry is widely used to
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doi: 10.1016/j.jcpa.2004.04.003
Correspondence to: C. H. Liu, Department of Veterinary Medicine, National Taiwan University, No. 1, Section 4, Roosevelt Road, Taipei 106, Taiwan.
detect microbial antigen in tissue sections from naturally infected animals, except when specific primary antibodies are commercially unavailable or excessively expensive. In such cases, the use of positive antiserum from infected members of the homologous species is of potential valve. Although the use of homologous primary antiserum is not usually applicable in immunohistochemistry, a suitable method was recently described byLu and Partridge (1998). In the present study, an avidin-biotin-complex and alkaline phosphatase (ABC-AP) method was developed in which ELISA-positive murine antiserum was used as the primary antibody for the diagnosis of MHV and M. pulmonis infection.
Materials and Methods Animals
Experiment 1. Specific-pathogen-free cAnNCrj.Cg-Foxn1nu/Foxn1nu and Foxn1nu/þ nude mice, orig-inally obtained from Charles River Laboratories (Yokohama, Kanagawa, Japan), were maintained at the National Laboratory Animal Center (NLAC) in Taiwan. Thirty of these mice, consisting of 20 homozygous males aged 5 weeks and 10 homo-zygous females aged 9 weeks were sold, to a unit that maintains animals for use in research in Taipei City, on February 2 and March 8, 2000, respectively.
Animals were housed in sterile microisolator cages (Laboratory Products, Maywood, NJ, USA), kept in animal cabinets (Nu-air; Plymouth, MN, USA), and placed in conventional rooms. Sterile water and commercial rodent chow (PMI Feeds, St Louis, MO, USA) was provided ad libitum. Bedding changes, water replenishment and supply of food were carried out in a class-II biological safety cabinet. The animal houses were maintained at 20 to 25 8C with a 12-h light/dark cycle.
At 42 – 49 days after arrival at the unit, the male nude mice had become emaciated, anorexic and dehydrated, with scaly skin, hunched posture, diarrhoea and ocular discharge, and 50% had died. The female nude mice showed similar but less severe signs. Four male and two female nude mice were humanely killed at the age of 15 – 16 weeks and samples were taken back to the NLAC for pathological examination.
Experiment 2. Sixty 6-week-old female Wistar rats from the NLAC were transferred to a clean conventional local area and housed in autoclaved microisolators. Bedding was changed twice a week in class II biohazard cabinets. Animals were main-tained at a room temperature of 20 – 25 8C and humidity of 50 – 70%, with a 12-h light /dark cycle.
Sterile water and commercial rodent chow (PMI Feeds) were provided ad libitum. Bedding (Beta-chip; Northeastern Inc., Warrensburg, NY, USA), polysulfone cages (Laboratory Products) and supplies were all autoclaved. A proportion (30%) of the animals showed sneezing 5 – 6 weeks after arrival. Thirteen of the affected animals, now aged 12 – 24 weeks, were killed for diagnostic evaluation.
Necropsy revealed consolidation of the apical and cardiac lobes of the lungs. ELISA screening for pneumonia virus of mice (PVM), Sendai virus, lymphocytic choriomeningitis virus (LCMV), and sialodacryoadenitis virus (SDAV) was negative. Anti-body titres of three rats for M. pulmonis, assessed by ELISA score, were 6.07 to 17.22. ELISA results were interpreted on the basis of the method described below.
Preparation of ELISA-positive Sera
The ELISA monitoring programme and diagnostic service of the NLAC were used to detect the following infectious agents: pneumonia virus of mice (PVM), reovirus (Reo-3), Sendai virus, lym-phocytic choriomeningitis virus (LCMV), Theiler’s murine encephalomyelitis virus (GD VII), minute virus of mice (MVM), mouse hepatitis virus (MHV), mouse adenovirus (Mad), ectromelia virus, Kilham’s rat virus (KRV), sialodacryoadenitis virus (SDAV/RCV), M. pulmonis, hantavirus, K virus, and Clostridium piliforme. The procedure followed the Charles River ELISA scoring system (Serology Method Manual; Charles River Laboratories, Wilmington, MA, USA). Briefly, 50 ml of serum sample, diluted 1 in 60 in BLOTTO (Bovine Lacto Transfer Technique Optimizer; 5% non-fat dry milk in phosphate-buffered saline (PBS)) (Johnson et al., 1984), were added to each appropriate antigen well and control well. The plate was covered and incubated for 40 min at 37 8C. After several wash-ings, 50 ml of horseradish peroxidase-conjugated, affinity-purified horse anti-mouse or anti-rat IgG (Kirkegaard and Perry Laboratories, Maryland, USA), depending on species, were added to each well. After incubation for 40 min at 37 8C, the plate was washed again. One hundred ml of 0.4 mM ABTS-2.0 mM H2O2 chromogenic substrate were then added to each well and the plate was incubated at room temperature for 40 min. Absor-bance was determined colorimetrically at 405 nm with an ELISA microplate reader (Thermo-max;
Molecular Devices, Sunnyvale, CA, USA). Absor-bance values were transmitted from the ELISA reader to a personal computer (PC) where they were converted to scores by dividing by 0.13.
The denominator of 0.13 divided net absorbance values of 0.13 to 1.3 into scores of 1 to 10. Integer scores were read and interpreted by comparison with the 3-decimal absorbance values. The PC was also used to compute net scores (Scoreantigenminus Scoretissue control). A result was considered non-specific and recorded as tissue control (TC) when both Scoreantigenand Scoretissue controlwere 2 or above. Provided that the Score tissue control was lower than 2 (absorbance values lower than 0.26), net scores were interpreted as follows: 0 – 1, nega-tive; 2 – 3, borderline; $ 3, positive. When the test serum was interpreted as “single agent (i.e., MHV)-positive”, and the net score was $ 10, serum samples were collected and stored at 2 20 8C until used as primary antibody for immunohistochemistry.
Pathological Examination
The lungs, trachea, lymph nodes, heart, liver, spleen, small intestine, stomach, kidneys, urinary bladder, adrenal glands, skin and brain from affected animals in experiments 1 and 2 were fixed in 10% neutral buffered formalin. The tissue samples were processed by routine methods to paraffin wax-embedded blocks. Sections (6 mm) were stained with haematoxylin and eosin (HE).
Immunohistochemistry
The Mouse on Mouse kit (M.O.M.e; Vector Laboratories, Burlingame, CA, USA) was used for the immunohistochemistry study of MHV infection in experiment 1. The primary antibodies were ELISA-positive, mouse anti-MHV sera (ELISA score; 11.2 – 14.4). Infection with PVM, Reo-3, Sendai virus, LCMV, GD VII, MVM, Mad, ectrome-lia virus, K virus, M. pulmonis and polyoma virus were ruled out on the basis of ELISA results.
Tissue sections were dewaxed in xylene and rehydrated in a graded alcohol series. Antigen unmasking was performed by immersion of sec-tions in Vector antigen unmasking solution 1% in PBS and boiling for 5 min in a 1450-W microwave oven (RE-C102; Sampo Co., Taiwan). The sections were then immersed in cool PBS for 10 min, rinsed in PBS, and incubated with trypsin (Sigma Chemi-cal Co., St Louis, MO, USA) 0.1% in PBS for 5 min at 40 8C. Endogenous peroxidase activity was quenched with hydrogen peroxide 0.3% in metha-nol for 5 min at 40 8C. The sections were then rinsed in PBS, incubated for 30 min at 40 8C in mouse M.O.M.e IgG blocking reagent, rinsed with PBS, and incubated in M.O.M.e diluent for 5 min at 40 8C. Subsequently, they were incubated in
ELISA-positive murine anti-MHV serum (diluted 1 in 60 in M.O.M.e diluent) as the primary antibody for 24 h at 4 8C. Substitution of PBS or mouse serum (negative for any pathogen) for the primary antibody served as a negative control. The sections were then rinsed in PBS, incubated with M.O.M.e biotinylated anti-mouse IgG reagent for 60 min at 40 8C, rinsed in PBS, incubated in Vectastainw ABC-AP reagent for 60 min at room temperature, rinsed in PBS, and incubated with alkaline phos-phatase substrate solution (Vectorw Red; Vector Laboratories) in 100 mM Tris HCl, pH 8.2 – 8.5, for 30 min at room temperature. Endogenous alkaline phosphatase activity of tissues was inhibited by adding one drop of levamisole (Vector Laboratories) to 5 ml of Tris HCl buffer before preparation of the substrate working solution. The sections were rinsed in distilled water, counter-stained with Mayer’s haematoxylin and examined microscopically.
Lung sections from experiment 2 were pre-treated as described in experiment 1. They were then rinsed in PBS, incubated for 30 min at 40 8C in diluted (1 in 20) normal horse serum (Vector Laboratories), incubated with diluted (1 in 60) ELISA-positive murine anti-M. pulmonis serum (ELISA score:13.4 – 17.7) as primary antibody for 24 h at 4 8C, rinsed in PBS, and incubated with diluted biotinylated horse anti-mouse IgG second-ary antibody (1 in 100, rat-absorbed) for 60 min at 40 8C. The subsequent procedures were the same as those in experiment 1.
Results ELISA Monitoring Results
In the MHV test, 485 mouse serum samples were examined. Of these, 465 were negative (score , 3), three were positive (score 3 – 10), and 17 had a high MHV score (. 10). Seven of 17 samples with a high score were excluded because of simultaneous positivity for GDVII. Ten serum samples (ELISA score: 11.2 – 14.4) were collected for further diag-nosis of MHV infection in experiment 1.
In the M. pulmonis test, 268 rat serum samples and 383 mouse serum samples were examined.
Only six rat samples were positive, and 380 mouse samples were negative (score , 3). Three mouse samples showed a high M. pulmonis score (. 10), but one of these was excluded because of simul-taneous positivity for MHV. The remaining two mouse serum samples (ELISA score: 13.4 – 17.7) were used for the diagnosis of M. pulmonis infection in Wistar rats in experiment 2. These Wistar rats
were confirmed as having M. pulmonis infection by testing 13 rat serum samples; three rats (23%) had an ELISA score of 6.07 – 17.22, and four (31%) had typical pulmonary lesions.
Histopathology and Immunohistochemistry
Experiment 1. Gross examination of the four affected male nude mice revealed that the liver was firm and pale with multiple, white, depressed foci, 2 to 3 mm in diameter. Microscopically, necrotic foci were seen to be scattered throughout the hepatic parenchyma, being well demarcated from the adjacent normal tissues (Fig. 1). Multinucleated syncytial giant cells with basophilic cytoplasmic granules (20 mm in diameter) were often present.
Colonic and caecal mucosal ridges were attenuated and shortened, and contained syncytial cells (Fig. 2). Immunohistochemical results revealed
MHV antigen at the periphery of the necrotic foci in the liver (Fig. 3) and within the caecal and colonic multinucleated syncytial cells (Fig. 4), spleen, and jejunal and gastric mucosa.
Experiment 2. Microscopical examination of two affected Wistar rats showed extensive consolidation of the lung, with variable degrees of hyperplasia and metaplasia of bronchiolar epithelial cells, and mononuclear cell infiltration into the bronchioles and adjacent alveolar spaces. Loss of cilia and flattening of epithelial cells in the bronchioles were noted (Fig. 5). Aggregates of necrotic debris and neutrophils were present in the bronchiolar lumina. Peribronchial and perivascular cuffing by lymphocytes, macrophages and plasma cells was also observed. Sections of lung showed immunohistochemical labelling for M. pulmonis
Fig. 1. MHV-infected mouse liver showing necrosis with multi-nucleated syncytial giant cells (arrow) at the periphery of the lesion. HE. Bar, 50 mm.
Fig. 2. Syncytial cells (arrowhead) in the surface epithelium of the colon of a MHV-infected mouse. HE. Bar, 25 mm.
Fig. 3. MHV antigen within necrotic hepatocytes and syncytial cells (arrowhead) immunolabelled with ELISA-positive serum as primary antibody. ABC-AP with haematoxylin counterstain. Bar, 25 mm.
Fig. 4. Colonic epithelial and syncytial cells (arrowheads) immunolabelled for MHV with ELISA-positive sera.
ABC-AP with haematoxylin counterstain. Bar, 25 mm.
antigen over the luminal surface of hyperplastic bronchiolar epithelial cells (Fig. 6).
Discussion
Only 10 serum samples with a positive ELISA titre for MHV alone and two with a positive titre for M. pulmonis alone were used in this study. The results indicated the applicability of the technique to diagnosis in the absence of access to commer-cially produced antibodies. There have been few reports of the use of ELISA-positive murine sera as primary antibody for the immunohistochemical examination of rodent tissue. Polyclonal antiserum has been used for the diagnosis of MHV and M. pulmonis infection (Brownstein and Barthold, 1982; Brunnert et al., 1994; Liang et al., 1995). MHV
is the most common viral pathogen of mice, and seropositivity for MHV had been reported in 19 – 83% of animals in mouse colonies (Kraft and Meyer, 1986, 1990; Casebolt et al., 1988; National Research Council, 1991). In Taiwan, MHV is highly prevalent in mouse colonies, especially in immu-nosuppressed mice (Liang et al., 1995). Due to the inability of immunodeficient homozygous mice to produce antibody, immunohistochemistry may represent a useful replacement for serology in diagnosing MHV infection. In this study, high-titre serum from immunocompetent mice was used as primary antibody. The findings regarding the distribution of MHV antigen in experiment 1 accorded with those of previous reports (Weir et al., 1987; Barthold et al., 1990), the antigen being demonstrated in the intestine, liver, spleen and stomach, consistent with multi-organ MHV infection (Compton et al., 1993).
Serology and culture are widely used in the diagnosis of M. pulmonis infection, but discrepan-cies sometimes occur (Cassell et al., 1981). ELISA has detected M. pulmonis infection in 8 – 78% of rat colonies and 35 – 91% of mouse colonies, depend-ing on whether conventional or barrier-maintained facilities are used (Casebolt et al., 1988; Kraft and Meyer, 1990). An advantage of ELISA is the low incidence of non-specific or false positive reactions as compared with haemagglutination inhibition (HI) (Kraft and Meyer, 1986). Discrepant results for M. pulmonis infection obtained by different serological tests may be due to reactive substances in the serum, such as lysozyme, antinuclear antibodies, protease and bacterial products (LaRegina et al., 1987). Culture of M. pulmonis from tracheobronchial lavage fluid showed 89.6%
positivity in rats and 36.5% positivity in mice in non-barrier-maintained facilities (Timenetsky and DeLuca, 1998). For routine monitoring of M. pulmonis, the preferred use of time-consuming culture procedures as opposed to serological testing is applicable only in acute or early infection.
One-third of infected animals do not yield M. pulmonis in culture (Kraft et al., 1982). Culture and histopathology may be misleading in evaluat-ing a colony of rodents for mycoplasma infection, particularly when the prevalence is low (Cox et al., 1988).
M. pulmonis infection in the chronic stage is readily detected histopathologically (Kraft et al., 1982; Goto et al., 1994), but in some instances MHV or mycoplasmal infection produces minimal or no lesions. In such instances, immunohistochemistry is valuable (Matthaei et al., 1998). In experiment 2, labelling of M. pulmonis antigen was noted on
Fig. 5. Hyperplasia of bronchiolar epithelial cells of a rat infected with M. pulmonis. HE. Bar, 25 mm.
Fig. 6. M. pulmonis antigen is clearly demonstrated on the luminal surface of hyperplastic bronchiolar epithelial cells. ABC-AP with haematoxylin counterstain. Bar, 25 mm.
the luminal surface of bronchiolar epithelial cells, a site also affected by the cilia-associated respiratory (CAR) bacillus (Matsushita et al., 1987). In a previous study (Brunnert et al., 1994) the ABC method failed to detect M. pulmonis in formalin-fixed lung tissue but gave 27.4% (17/62) positive results with ethanol-fixed lung tissue; this com-pared with 96% (60/62) positive results given by the polymerase chain reaction (PCR). However, MHV antigens were demonstrated in formalin-fixed, paraffin wax-embedded blocks for as long as 2 years after preparation (Brownstein and Barthold, 1982). Immunohistochemistry, com-monly used to detect rodent pathogens, takes much less time than that required for culturing mycoplasmas. In the present study, however, the incubation with primary antiserum was carried out overnight at 4 8C (Miller and van der Maaten, 1989), rather than for 30 min at room temperature or at 40 8C (Liu et al., 1997; Liang et al., 2000), the purpose being to increase the immunolabelling.
PCR is more sensitive than immunohistochem-istry or microbial isolation but requires a high degree of technical expertise, and contamination leads to false positivity (Brunnert et al., 1994). It offers an advantage over serological testing, how-ever, in situations in which an antibody response is unlikely to be generated. In conclusion, the method described, in which ELISA-positive serum was used as primary antibody, may be useful in diagnosing infection in immunodeficient animals or when commercial immunohistochemical reagents are unavailable (Matthaei et al., 1998).
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