國立臺灣大學獸醫專業學院獸醫學研究所 博士論文
Graduate Institute of Veterinary Medicine
National Taiwan University Doctoral Dissertation
組織內病毒蛋白與核酸偵測之最佳化:鼠肝炎病毒與 犬瘟熱病毒
Optimization of the in situ detection of viral protein and nucleic acid in tissue: mouse hepatitis virus and canine
distemper virus 梁鍾鼎
Chung-Tiang Liang
指導教授：劉振軒 博士 闕玲玲 博士 Advisors： Dr. Chen-Hsuan Liu Dr. Ling-Ling Chueh
中華民國 100 年 6 月, June, 2011
致謝
人生每一次的「起｣都是期待的開始，每一次的「落｣更是另一個期待的開始。
在這段博士研習期間，更是嘗盡了實驗失敗的挫折。人生將近五十個年頭，這本 博士論文的完成代表著我有更多的責任，不只是對動物、家庭、科學、獸醫學及 實驗動物醫學。這一路走來，首先要感謝劉教授振軒對我博士論文的指導、一路 鼓勵我，讓我能一次再一次面對挫折，並有繼續走下去的勇氣；此外，他總是給 學生許多機會，在許多公開場合演講，建立自己的信心。接著我要感謝闕教授玲 玲，總是能一針見血看出論文的缺憾，給予直言建議；此外，她總是大方地分享 手邊的病例，使我能有眼界大開般的驚豔。同時要感謝梁主任善居、龐教授飛及 李主任進成這幾年給予學生的支持與關懷，讓我受益良多。
接著要感謝我的父母，雖然他們兩位已在 2007 年同一年內相繼往生，但是 也為自己這段期間忙於學業及工作，脾氣暴躁。未能於兩位老人家晚年，多陪陪 他們而深感愧疚。希望這小小的突破，能安慰他們兩位在天之靈。
我還要感謝我的內人春燕及子女博宏、洛涵，這將近九年的時光一路上的支 持、包容及默默付出，看著小孩由出生而漸漸長大，使我感到没好好陪你們而非 常抱歉。
同時要感謝其它師長及同事、同學們如 Dr.C Kai，黃博士彥智、張博士維 正、王博士雅芳、憲青、泔泓、宣憲、思偉、勝裕、宜興、益誠、秀瓊、秀鈴、
譯瑩、儼峰兄、國亨兄、與我同星座的余副主任俊強、蘇教授璧伶、鄭教授謙仁 等在臨床病理診斷、分子診斷、切片染色、原位雜交染色、病毒分離、及 H 基因 型樹狀圖分析等實驗給予的協助。
最後要感謝 2010 年在美國馬里蘭州巴爾的摩市約翰霍布金斯醫學院(JHU) 分子及比較病理系接受病理獸醫師訓練五個月期間；Dr.C Brayton，Dr.SL Poynton，Dr.KL Gabrielson，Dr.RJ Montali 等教授給予的指導與照顧，...
族繁不及備載，感謝過程中所有的人、事、物、國研院國家實驗動物中心經費支 援及梁主任給我這機會在職進修，當然還有往生的小狗狗及小老鼠們。謝謝大 家！
I
Contents
Abstract II
中文摘要 V
Chapter I
General Introduction 1Chapter II
Chapter III
Immunohistochemical diagnosis of mouse hepatitis
virus and Mycoplasma pulmonis infection with murine antiserum
J Comp Pathol 131: 214–220, 2004
A non-biotin polymerized horseradish - peroxidase method for the immunohistochemical diagnosis of canine distemper
J Comp Pathol 1 13 36 6: :5 57 7- -6 64 4, , 2 20 00 07 7
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36
Chapter IV
Improving detection of canine distemper virus in formalin-fixed, paraffin-embedded, tissues: using in situ hybridization with integrated optical density to give a semi-quantitative assessmentManuscript in preparation
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Chapter V
Phylogenetic analysis and isolation of canine distemper viruses in TaiwanTaiwan Vet J 34(4): : 198-210, 2008 (
台灣獸醫誌2009
年度優良論文獎)
73
Chapter VI
Canine Distemper in Taiwan from 2000 – 2009:co-infections, and use of RT-PCR and immunohistochemistry to detect tissue involvement in two groups of dogs
J Appl Res Vet Med,2011 ( (s su ub bm mi it tt te ed d) )
87
Chapter VII
General Discussion 125Appendix
Curriculum Vitae
136 143
II
Abstract
Mouse hepatitis virus (MHV) is the leading viral pathogen of laboratory mice in Taiwan. This study established a modified alkaline phosphatase-labelled avidin-biotin-complex (ABC-AP) method for detection of MHV in tissues. Mouse hepatitis virus antigen was clearly detected in samples of liver, stomach, caecal and colonic mucosa, and spleen. This method may prove useful in diagnosis when commercial antisera are unavailable or when immunosuppression prevents serological diagnosis. (Chapter II). Canine distemper virus (CDV) causes a highly contagious disease, which has been reported in Taiwan for many years; however the causative and its genes have never been identified and pathogenesis are poorly understood. The objectives of the dissertation were to set up a fast and easy diagnostic method of CDV infection, to isolate the field virus and do phylogenetic analysis of the viral H gene, to characterize the pathology of CD in Taiwan, and to assess the frequency of CNS demyelination and other pathological lesions in cases of CDV infection confirmed by immunohistochemistry (IHC) and/or reverse transcription polymerase chain reaction (RT-PCR). This study describes a modified non-biotin polymerized horseradish peroxidase (HRP) immunohistochemical method for the diagnosis of CDV infection from formalin-fixed, paraffin-embedded tissues. This method confirmed seven out of eight (87.5%) suspected cases. Labeled CDV antigen was observed in cerebrum, cerebellum, meninges, glial cells, neurons, vascular endothelium, periventricular areas and pericytes, and choroid plexus; grey and white matter and central canal of the spinal cord; renal pelvis and tubular epithelium, and urinary bladder epithelium;
macrophages and lymphocytes in splenic white pulp and lymph nodes; skin epidermis;
bronchiolar epithelium and alveolar macrophages; hepatic Kupffer cells, and gastric and intestinal mucosal epithelium; stratified squamous epithelium of the tongue and oesophagus.With the non-biotin HRP detection system, pretreatment by autoclaving followed by microwave heating gave better labeling results than did microwave pretreatment alone. No obvious difference was noted between the labeling results produced by the non-biotin HRP detection system and the Super Sensitive TM Link-Label IHC detection system (Chapter III, VI). For the purpose of confirming the IHC labeling and improving the detection of CDV nucleoprotein RNA, in situ hybridization (ISH) was applied in paraffin-embedded tissues from selected dogs with spontaneous CDV infections. In addition to proteinase K, autoclaving in various solutions (Trilogy, TBS S3006, H-3301, and S1700) for pre-treatments were compared. The intensity was assessed by using the integrated optical density (IOD) and the integrity of the tissue morphology. The combination of proteinase K digestion and autoclaving in a Trilogy solution resulted in a 5- to 100-fold ISH signal enhancement of CDV RNA. This modified technique can be useful in the
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retrospective viral studies across a broad range in the future (Chapter IV). For the purpose of CDV genotyping, during the period from 2003 to 2005, two CDV strains were isolated from 17 non-vaccinated puppies with suspected canine distemper by co-culture with peripheral blood mononuclear leucocytes and B95a cells. In addition, four cloned hemagglutinin (H) genes were obtained from 166 dogs infected with CDV.
Indirect immunofluorescence assays and antigen tests confirmed that they were CDV.
Analysis of the H genes of the six identified strains revealed that the deduced amino acid sequences contained nine potential sites for N-linked glycosylation, as had been found for the H proteins of Japanese isolates. The seventh site is characteristic to the Taiwan strains described in this report and to the recently reported Japanese strains.
Furthermore, phylogenetic analysis of the H gene showed that the six isolates belong to the Asia-1 group and are closely related to the recently reported Japanese and Chinese strains (Chapter V). To realize the histopathological lesions of CDV in Taiwan, fifty two (IHC or RT-PCR positive) affected dogs were obtained from either animal clinics or dog shelters from 2000 to 2009, within which 32 were from clinics and 20 were from shelters. Postmortem and laboratory examination included, gross findings, histopathology, Luxol-fast blue cresyl echt violet (LFB-CEV) histochemistry, non-biotin HRP anti-CDV immunohistochemistry, and phosphoprotein gene RT-PCR.
Clinic cases had histories of treatment and or vaccination. Twenty four clinic cases (75%) were puppies less than 6 months old. Seventeen shelter cases (85%) were identified as ‘adults’ greater than 6 months old. There were 27 males and 25 females.
Eleven dog breeds were represented, but most dogs (35/52, 67%) were crossbred.
Totally, 79% (41/52) showed lymphoid depletion, 71% (37/52) had interstitial pneumonia, 65% (34/52) had CNS demyelination, 32% (17/52) had catarrhal enteritis.
Younger dogs from clinic group frequently had lymphoid depletion (31/32, 96%), inclusion bodies (28/32, 87%), pneumonia 81% (26/32, 81%), and CNS demyelination (26/32, 81%), which were all statistically significantly different from those from shelter. Enteritis was identified in about one third of the animals in both groups. The distribution of inclusion bodies also showed significant difference in urinary bladder, lymphoid tissues, lung, and alimentary tract between the two groups.
Twenty nine co-infections and other associated lesions were identified. However, no significant difference was seen in the frequency of occurrence between two groups with the exception of interstitial nephritis. In conclusion, lymphoid depletion, pneumonia, and CNS demyelination were the most common CDV-infected principal lesions, and the inclusion bodies had a high occurrence in lymph nodes, spleen as well as mucosa epithelium of lung, stomach, urinary bladder and kidney (Chapter III, VI).
In the present study, it has been demonstrated that CDV in Taiwan has at least one clade of the phylogenetic tree showing more than 95% amino acid similarity (< 5%
IV
amino acid variation) in the H gene. The modified non-biotin polymerlized horseradish peroxidase (HRP) IHC labeling and ISH method are easy, optimal and accurate for retrospective diagnosis of CDV and MHV. The occurrence of CDV-associated pathological lesions depend on the source of the animals, treatment history, age and tissue distribution.
Key word: mouse hepatitis virus; canine distemper virus; immunohistochemistry;
reverse transcription polymerase chain reaction; in situ hybridization
V
中文摘要
鼠肝炎病毒（mouse hepatitis virus；MHV）是小鼠族群高度傳染性首要病 原。本研究在缺乏特異性冠狀病毒初級抗體情況下，利用血清酵素免疫吸附法
（ELISA）鼠肝炎病毒陽性檢體血清，結合卵白素(avidin)與生物素化
(biotinylated)的二級抗體，在發病免疫缺陷鼠之福馬林固定組織，如肝、胃、
脾臟、 盲腸、及結腸等偵測鼠肝炎病毒抗原存在(Chapter II)。犬瘟熱病毒
（canine distemper virus, CDV）為一年代久遠的疾病，本病之發生，己遍及 全世界。犬瘟熱病毒為一高傳染性疾病，且存在臺灣許多年。然而；犬瘟熱病毒 分離，基因型卻不曾有報告。組織病變，免疫化學染色診斷，病理學分析及併發 症等均缺乏全面性調查資料。本研究目的為建立快速及正確的犬瘟熱病毒感染診 斷方法，分離犬瘟熱病毒株及分析其H基因之遺傳差異，及經免疫化學染色(IHC) 或反轉錄聚合酶反應(RT-PCR)確診下之自發病例，分析其中樞神經脫髓鞘發生率 及其它病理變化。研究發現經非生物素性聚合山葵過氧化酶 (non-biotin HRP) 免疫化學染色方法，對福馬林固定、石蠟包埋組織之犬瘟熱感染病例，其診斷率 可達87.5%。犬瘟熱病毒特異性抗原可在下列組織發現：大腦皮層外錐狀細胞及 樹狀突細胞質、小腦白質層星狀細胞及細胞質、軟腦膜、神經膠質細胞、神經元、
血管內皮細胞、腦室周圍、室管膜細胞、脈胳叢；脊髓灰、白質及中央導水管；
腎盂上皮及腎小管上皮細胞；膀胱黏膜上皮細胞；脾臟白髓及淋巴結巨噬細胞及 淋巴球；皮膚海綿層細胞及細胞質；肺臟小支氣管黏膜上皮及肺泡巨噬細胞；肝 臟巨噬細胞；胃腸黏膜上皮細胞；扁桃腺及食道複層扁平上皮細胞等。組織經高 溫高壓滅菌鍋前處理的免疫化學染色方法，其抗原復舊程度遠遠超過傳統微波爐 法，大大提高犬瘟熱病毒診斷率(Chapter III,VI)。為了要確認免疫化學染色訊 號，本研究建立一個改良的原位雜交染色步驟，應用於上述病例。此方法除了傳 統的蛋白水解酶K的雜交前處理外；另外比較了將切片置入於不同(Trilogy, TBS S3006, H3301, S1700)溶液中進行高溫高壓滅菌鍋前處理。同時原位雜交訊號使 用平均光密度(IOD)及組織形態完整性進行半定量性效果分析。結果發現進行原 位雜交前，切片如果配合蛋白質水解酶K先處理，再以Trilogy溶液高溫高壓處理 後進行原位雜交，其訊號及組織完整性均較其它組強，此一方法建立，未來將廣 泛應用於犬瘟熱病例或其它病毒性疾病之原位雜交診斷(Chapter IV)。為了解台 灣地區犬瘟熱病毒基因型分析，2003-2005年間進行病毒分離，自17隻未經疫苗 注射之發病幼犬，應用患犬血液單核球與 B95a 細胞株共同培養之技術，分離出 兩株具誘發融合細胞病變之病毒，經免疫螢光染色及抗原測試確認為犬瘟熱病 毒。將此兩株病毒之血球凝集素基因（H），與同期間自台灣大學動物醫院臨床 送檢病例之另外4株犬瘟熱病毒之H基因進行核酸定序。經比對序列與樹狀圖分析 發現，本土病毒株皆有9個N連結配醣位，其中第7個配醣位為日本或中國大陸流 行之亞洲1型犬瘟熱病毒所特有。本研究顯示臺灣地區所流行之犬瘟熱病毒，其H 基因經分析屬亞洲1型 (Chapter V)。為了解台灣地區是否有犬瘟熱病毒感染之
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病理變化差異，2000-2009年分析來自收容所或臨床動物醫院之犬瘟熱患犬計52 隻(經IHC或RT-PCR確診)。診斷方法包括肉眼檢查、組織病理、脫髓鞘特殊染色 (LFB-CEV)、IHC、RT-PCR等。結果發現32隻來自臨床動物醫院，20隻來自收容所。
來自臨床動物醫院之犬，75%為6月齡或以下，但來自收容所之犬，85%為大於6 月齡。共計11種品種，但67% 為混種犬。在病理學分析，淋巴減少(79%)、間質 性肺炎(71%)、中樞神經脫髓鞘(65%)、卡他性腸炎(32%)為主要病變。臨床動物 醫院組相對地在淋巴減少(96%)、病毒包涵體(87%)、間質性肺炎(81%)及中樞神 經脫髓鞘(81%)發生率很高，且統計差異顯著。腸炎占二組別之30-34%，無統計 差異顯著。犬瘟熱病毒病毒包涵体在膀胱、淋巴組織、肺臟及消化系統，二組別 有顯著差異。在29種犬瘟熱病毒感染之併發症或協同病變分析中，除了間質性腎 炎外，二組間皆無顯著性統計差異。總結而言，淋巴減少、肺炎、中樞神經脫髓 鞘是主要病變，然而病毒包涵體出現却以淋巴組織及黏膜上皮如腎臟、膀胱、肺 臟及消化系統較高(ChapterIII,VI)。綜合以上結果，本研究證明台灣犬瘟熱病 毒至少存在一種以上H基因型(亞洲1型)，其彼此間的胺基酸差異小於5%。同時本 研究所建立的免疫化學染色及原位雜交染色方法使回溯性犬瘟熱病毒及鼠肝炎 病毒之研究及診斷更簡單及正確。犬瘟熱病毒分離之方法可應用於未來臺灣不同 地區之病毒分離。犬瘟熱病毒感染之伴隨病變視動物來源、治療病史、年齡及組 織分布而定。
關鍵字: 鼠肝炎病毒; 犬瘟熱病毒; 免疫化學染色; 反轉錄聚合酶反應; 原位 雜交
1
Chapter I
General Introduction
2
1.1. Background
A critical factor for both in situ hybridization (ISH) and immunohistochemistry (IHC) detection of viral protein and nucleic acid is fixation. The optimal and best fixatives for tissue were either paraformaldehyde, low concentrationof formalin (5%
of formalin) (Yan et al., 2010) or 10% NBF, zinc formalin and alcoholic formalin (Babic et al., 2010).In brief, all aspects of tissue processing, including time until tissue fixation, type of fixative, duration of fixation, post-fixation treatments, and sectioning of the sample, impact the staining results (Babic et al., 2010). However, the tissue fixative formaldehyde initiates DNA denaturation (interchain hydrogen bonds break and bases unstack) at the AT-rich regions of double-stranded DNA creating sites for chemical interaction (Srinivasan et al., 2002; Shi et al., 2001). In this case, some heat-induced antigen retrieval (HIAR) pretreatment of the histological slides can enhance the signal of IHC or optimize the detection effect of nucleic acid by ISH (Srinivasan et al., 2002; Shi et al., 2001).
Canine distemper (CD), which is caused by a morbillivirus genus, family Paramyxoviridae, produces systemic or central nervous system (CNS) infections of dogs and related species and it is associated with high mortality in Taiwan (Wu et al., 2000; Yu et al., 2001). It is a non-segmented, single–stranded negative RNA virus of
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one envelope-associated protein (M), two glycoproteins (the haemagglutinine/attachment protein H and the fusion protein F), two transcriptase-associated proteins (the phosphoprotein P and the large protein L), and the nucleo-capsid N that encapsulates the viral RNA (Sidhu et al.,1993).
Diagnostically, CDV is typically detected following either immunohistochemical labelling (Frisk et al., 1999; Engelhardt et al., 2005; Liang et al., 2007), reverse transcription-polymerase chain reaction(RT-PCR) (Frisk et al., 1999), a sample antigen test (Liang et al., 2007), histopathological detection of intranuclear and cytoplasmic inclusion bodies, demyelination of cerebellar and cerebral white matter (Koutinas et al., 2002; Vandevelde and Zurbriggen, 2005; Kubo et al., 2007; Sips et al., 2007), footpad hyperkeratosis (Engelhardt et al., 2005; Koutinas et al., 2004;
Okita et al., 1997), immunofluorescence (Engelhardt et al., 2005), clinicopathological findings (Amude et al., 2007) or a combination (Frolich et al., 2000).
The characteristic CNS changes of CDV infection include polioencephalomacia, white matter demyelination, astrogliosis, eosinophilic intranuclear and cytoplasmic inclusion bodies, gemistocytes, and occasional multinucleated syncytial giant cells (Summers et al., 1984; Summers and Appel, 1985). However, syncytial giant cells were demonstrated in only 9 - 28 % of CDV-infected brains (Palmer et al., 1990; Wu
4
% (Haines et al., 1999), 33% (Stanton et al., 2002), 38.1% (Wu et al., 2000) or 72%
(Palmer et al., 1990). Thus, histological examination of lesions does not appear to be a reliable indicator of CDV infection.
1.2. Pathogenesis of canine distemper virus infection
The pathogenesis and clinical features have been widely reported (Appel, 1970;
Beineke et al., 2009). Great variations in duration, severity and clinical presentation of distemper have been found in experimentally infected dogs as well as in animals suffering from this world-wide spontaneously occurring disease. The incubation period may vary from 1 to 4 weeks and depends on the viral strain, age of the animal at the time of infection, and immune status of the host. Disease manifestation ranges from virtually no clinical signs to severe disease with approximately 50% mortality.
The virus is shed primarily by oro-nasal secretion. Tissue macrophages and monocytes located in or along the respiratory epithelium and tonsils represent the first cell type to pick up and propagate the virus. Following this local burst of virus replication, the pathogen is then disseminated by lymphatics and blood to distant hematopoietic tissues during the first viremic phase. However, its CNS pathogenesis is not completely clarified. In the early stage of the infection, demyelination is
5
demyelination is due to a bystander mechanism resulting from interactions between macrophages and antiviral antibodies (Beineke et al., 2009; Vandevelde and Zurbriggen, 2005).
1.3. Pathogenesis of mouse hepatitis virus infection
Mouse hepatitis virus (MHV) which is caused by an enveloped positive stranded coronavirus genus, family Coronaviridae, causes a variety of diseases, such as enteritis, hepatitis, in susceptible rodents (Haring and Perlman, 2001). Enterotropic
MHV strains, such as MHVY, initially replicate in epithelial cells of the
gastrointestinal (GI) tract after infection of adult immunocompetent mice. Disease is
acute and mild with minimal pathologic changes, and virus rarely disseminates to
other organs. In contrast, morbidity and mortality are high in neonatal
immunocompetent mice, and infection of immunocompromised mice can cause
multisystemic, persistent infection with extended viral shedding and high mortality.
Polytropic MHV strains, such as MHV-A59 or MHV-JHM (JHMV), initially replicate
in the proximal respiratory tract epithelium after infection, then disseminate to many
organs via viremia, lymphatic spread, or olfactory pathways from the nose to the brain.
Infection of adult immunocompetent mice results in subclinical infection, hepatitis,
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disseminated disease with high mortality (Compton et al., 1993; Liang et al.,1995).
The demyelinating JHM strain of MHV similar as CDV, infection of immunocompetent mice with the results in acute encephalitis, followed by the development of chronic demyelination in survivors (Haring and Perlman, 2001;
Weiner et al., 1973). The pathogenesis of Neurotropic JHM strain is dependent upon viral dose, route of infection, the host’s age and genetic background; however, prominent CNS infections can be induced with the neurotropic MHV strains either via the intranasal route or by direct inoculation into the CNS. The parental JHMV strain infects astrocytes, oligodendroglia, microglia and neurons (Stohlman and Hinton, 2001).
1.4. Objectives of the studies
The objectives of the studies were to set up an accurate and optimal IHC or ISH detection method of CDV and MHV infection, to isolate the field virus and do phylogenetic analysis of the viral H gene, to characterize the pathology of CD in Taiwan, and to assess the frequency of CNS demyelination and other associated lesions in cases of CDV infection confirmed by immunohistochemistry (IHC) and/or RT-PCR.
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1.4.1. Immunohistochemical detection of mouse hepatitis virus infection
Mouse hepatitis virus (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;
Liang et al., 2009). In Taiwan, MHV is highly prevalent in mouse colonies, especially in immunosuppressed mice (Liang et al., 1995; Liang et al., 2004). 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 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).
1.4.2. Immunohistochemical detection of canine distemper virus infection
Immunohistochemical detection of CDV antigen in tissue sections was reported by Palmer et al. (1990), to be superior to the demonstration of inclusion bodies or syncytial cells for the diagnosis of canine distemper encephalitis. In the infected dogs,
8
ependymal cells, inflammatory macrophages, choroid plexus cells, and meningeal cells (Mitchell et al., 1991); in haired skin and footpad epithelium (Haines et al., 1999;
Koutinas et al., 2004); and in lung, spleen, kidney, and lymph nodes (Iwatsuki et al., 1995).
The traditional avidin-biotin complex (ABC) immunohistochemical labelling method has several disadvantages. First, avidin has a high isoelectric point. At pH 7.4, it is positively charged and tends to bind to certain proteins (Haines and Chelack, 1991). The hydrophobic and electrostatic characteristics of the avidin conjugates appear to play a role in the non-specific binding (Masuoka et al., 2002). Second, kidney, pancreas, liver, lymphoid tissues, and nervous system tissue may contain lectin-like, negatively charged, endogenous, biotin-rich material which may adhere non-specifically to the ABC, resulting in false positive reactions (True, 1990;
Ramos-Vara, 2005). Although the background can be greatly reduced by replacing avidin with streptavidin, background from endogenous biotin is still a problem with streptavidin methods (Ramos-Vara, 2005).
This report describes the development of a novel method of non-biotin chain polymer conjugate labeling combined with heat-induced epitope retrieval (Shi et al., 2001; Ramos-Vara, 2005; Liang et al., 2007) for CDV diagnosis.
9
1.4.3. In situ hybridization detection of canine distemper virus infection
In situ hybridization (ISH) can be used to detect CDV RNA in formalin-fixed,
paraffin-embedded tissues (Muller et al., 1995; Gaedke et al., 1997; Hoyland et al., 2003; Engelhardt et al., 2005; Vandevelde and Zurbriggen, 2005; D’Intino, et al., 2006). However, in a retrospective study of the detection of viral RNA in formalin-fixed, paraffin-embedded tissues, the results of the ISH optimization following proteolytic enzyme digestion and heat-induced antigen retrieval (HIAR) pre-treatment were conflicting (Mcquaid et al., 1990; Gaedke et al., 1997; Kim and Chae, 2003; Shi et al., 2001). Additionally, few reports have focused on the optimization of the protocol for the detection of CDV RNA. While both proteinase K and saponin treatment preserved the morphology of tissues equally well, treatment with 0.1% saponin resulted in a more robust ISH signal than the proteinase K treatment (Yamawaki et al., 1993). Gaedke (1997) reported that the treatment with buffers made with DEPC water before hybridization, the storage time in paraffin wax, or the length of time the tissue was incubated in the fixative had no effect on the ISH-based detection of CDV RNA. Additionally, proteinase digestion, sodium borohydrite, or boiling in citrate buffer in the microwave had no effect.
However, other reports have demonstrated that proteinase K and protease VIII
10
enzymes, either increasing the concentration or increasing the length of the incubation produced no increase in the ISH signal. An incubation of 5 min at RT with 0.5 mg/ml protease VIII and microwaving resulted in the optimal signal for the detection of measles virus (Mcquaid et al., 1990). Similarly, antigen retrieval using a thermocycler combined with proteinase K digestion can enhance the ISH signal for the detection of porcine circovirus 2 (Kim and Chae, 2003). Because CDV are RNA virus which offer several unique limit regarding their in situ detection. The detection threshold of ISH which is estimated to be about 10 copies per cell. RNA viruses often produce lower copy numbers when they infect a cell and need in situ pretreatment before ISH (Amaro Filho and a Nicol, 2010).
The aim of this study was to optimize the ISH protocol and provided a combined HIAR pretreatment for the detection of CDV RNA in formalin-fixed, paraffin-embedded tissues in retrospective studies.
1.4.4. Viral isolation and phylogenetic analyses of field canine distemper virus
Dogs can be protected from the infection by live attenuated vaccine. Nevertheless, increasing incidences of canine distemper in pups in suburb areas and in urban kennel shops have recently been noticed in spite of vaccination. Apparent failure in
11
which interferes with vaccine virus (Kai et al.,1993; Iwatsuki et al.,2000).
Consequently, there are similar problems in Taiwan. As another possibility, antigenic changes in the currently prevalent CDV, which the current vaccine does not give complete protection, are also speculated (Kai et al., 1993; Lan et al., 2006).
Characteristics of prevailing CDV in the field, however, have not been investigated in Taiwan due to difficulties in isolation of wild CDV from the field samples. The isolation of CDV was difficult (Imagawa et al., 1980; Metzler et al., 1984). Even using B95a cell, the viral isolation rate might be zero (0/32), However, that report used older dogs, from 3-month to 9-year-old as resource for viral isolation (Shin et al., 1995). CDV was rarely isolated from plain brain homogenates (Kimoto, 1986).
However, using canine kidney tissue system (Ho et al., 1979; Evans et al., 1991), and canine lung macrophage cultures have been employed for the isolation of CDV without losing its virulence, but the cells are often contaminated with bacteria (Appel et al., 1967). Bovine proliferative cells and peripheral blood macrophages were also tried for CDV isolation (Metzler et al.,1981). The avirulent CDV may grow well in kidney cells but poorly in macrophages, whereas the virulent CDV grow well in macrophages but poorly in kidney cells (Evans et al., 1991). Vero cells have been widely used for the isolation of CDV as well as measles virus (Metzler et al.,1981,
12
passages are required for virus isolation (Ho et al.,1979). Vero.DogSLAMtag cells developed cytopathic effect (CPE) as early as 24 hours after inoculation, but it requires specific transfection techniques (Seki et al., 2003; Lan et al., 2006). Virulent CDV replicates well in mitogen-stimulated canine or ferret lymphocytes (Ho et al., 1981; Appel et al., 1992). However, CPE is not produced in these cells, so that the cultures have to be co-cultured with other susceptible cells such as Vero cells after confirming the presence of virus antigens by immunofluorescent technique (Appel et al., 1992). With such primary cells, their preparation and possibility of contamination with other canine viruses make them impractical.
Considering these drawbacks in the use of primary cells and long passage time of Vero cells, using B95a cells of marmoset B lymphoid cell line, which have been reported to be highly susceptible hosts for the isolation of wild measles virus from patient’s materials (Kobune et al.,1989), and the canine distemper virus isolation (Kai et al., 1993; Mori et al., 1994; Imagawa et al.,1999). In this study, we succeeded in the isolation of CDV in Taiwan by using B95a cells and compared the H gene phylogenetic analyses with other reference isolates. Phylogenetic analyses place the field Taiwan CDV strains in the Asia-1 group (Liang et al., 2008). CDV isolates in Taiwan are all have 530 G, 549Y and 178A residues of Haemagglutinin(H) gene
13
1.4.5. Retrospective pathological analysis of canine distemper in Taiwan
We have noticed that in Taiwan, there have been changes in the suite of histopathological lesions seen in the prevalent infection of CDV since 2000; CNS signs are now marked and gastrointestinal involvement is rare (Liang et al., 2007).
Two distinct disease groups of CDV infection, “enteritis and non-enteritis”, have also been noted in Japan. The non-enteritis type of CD exhibited reduced epitheliotropism, might be the wild-type of CDV infection (Okita et al., 1997). In other reports also, the frequency of CNS findings seemed lower than what we were experiencing in Taiwan:
eg. syncytial giant cells in 9 % of the infected brains (Palmer et al., 1990), and eosinophilic intranuclear or cytoplasmic inclusion bodies in only 17-72 % (Haines et al., 1999; Palmer et al., 1990; Stanton et al., 2002; Wu et al., 2000).
In addition to the lesions of CDV itself, infected dogs may have a wide variety of concurrent infections, including canine adenovirus type 2 (CAV-2)(Chvala et al., 2007; Damian et al., 2005), coccidiosis (Headley et al., 2009), colibacillosis (Wada et al., 1996), cryptosporidiosis (Fukushima and Helman, 1984), parainfluenza viruses (Damian et al., 2005), Mycoplasma cynos(Chvala et al., 2007), toxoplasmosis (Moretti et al., 2006), Tyzzer's disease (Headley et al., 2009), documented in
14
case analysis, it is interesting to know concurrent infections with CDV and characteristics of CDV-associated lesions in different environment for appropriate management. Thus, we a retrospective study was conducted, for the 10 years from 2000 to 2009, to compare CDV histopathological lesions and complications in two groups of dogs in Taiwan, 32 dogs that had been treated in clinics and 20 dogs from shelters.
15
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Chapter II
Immunohistochemical diagnosis of mouse hepatitis virus and
Mycoplasma pulmonis infection with murine antiserum
Reprinted from JOURNAL OF COMPARATIVE PATHOLOGY, Vol.131, Author(s):
Liang, C. T., Wu, S. C., Huang, Y. T., Lin, Y. C. , Chang, W. J., Chou, J. Y., Liang , S.
C. , Liu, C. H. Title of article: Immunohistochemical diagnosis of mouse hepatitis
virus and Mycoplasma pulmonis infection with murine antiserum, Pages No. 214-220,
copyright (2004) with permission from Elsevier.
Immunohistochemical Diagnosis of Mouse Hepatitis Virus and Mycoplasma pulmonis Infection
with Murine Antiserum
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.
q2004 Elsevier Ltd. All rights reserved.
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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0021–9975/$ - see front matter q2004 Elsevier Ltd. All rights reserved.
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.
