Identification of Helicobacter DNA in feline pancreas, liver, stomach, and duodenum: comparison between findings in fresh and formalin-fixed paraffin-embedded tissue samples

The clinical significance of Helicobacter spp. in feline digestive organs needs to be evaluated and formalin-fixed and paraffin-embedded (FFPE) tissue samples provide an invaluable source for molecular studies. In this study, we performed a PCR assay to investigate the presence of Helicobacter DNA in digestive organs from seven cats and compared this occurrence in fresh and formalin-fixed and paraffin-embedded (FFPE) tissue samples from the same organs. The present study identified Helicobacter DNA in the pancreas, liver, stomach, and duodenum in fresh tissue samples but only in the stomach in FFPE samples. To our knowledge this is the first time that Helicobacter DNA have been identified in the feline pancreas. This study indicates that it is important to be aware of differences between results when analyzing FFPE samples compared to fresh tissue samples, especially regarding longer DNA fragments (>200 bp (base pairs)).     

Immunohistochemical detection of feline calicivirus in formalin-fixed, paraffin-embedded specimens.

An immunohistochemical technique was developed for detection of feline calicivirus (FCV) in formalin-fixed, paraffin-embedded cultured cells and tissues. Initial trials with cultured cells indicated that the indirect immunoperoxidase method using rabbit antiserum to FCV strain 255, and horseradish peroxidase-labelled antibodies to rabbit immunoglobulin G lacked sensitivity and showed excessive diffuse background staining despite trypsin digestion of sections before staining. An amplified indirect immunoperoxidase technique using commercially available biotinylated antirabbit antibodies and avidin-biotin-peroxidase or streptavidin-peroxidase (SP) complexes proved highly successful. When optimal conditions, including those for trypsinization, inactivation of endogenous peroxidase and blocking were determined, the SP technique was preferred. Applied to tissue of cats in the acute phase of FCV infection, the technique provided clear identification of cells containing FCV antigens in sections in which histological detail was well preserved.     

Identification of monoclonal antibodies for immunohistochemical staining of feline B lymphocytes in frozen and formalin-fixed paraffin-embedded tissues.

Commercially-available monoclonal antibodies to B lymphocytes were evaluated for immunohistochemical staining of feline B lymphocytes in frozen and formalin-fixed, paraffin-embedded tissues using an avidin biotin complex immunoperoxidase immunohistochemical technique. Three monoclonal antibodies: F46A and F72A raised to "carnivore" B lymphocytes and RA3.6B2 raised to murine B lymphocytes, stained B lymphocyte-dependent areas of frozen feline lymphoid tissue. In addition, antibody RA3.6B2 stained B lymphocyte dependent areas in formalin-fixed, paraffin-embedded feline tissues. There was no staining of T lymphocyte-dependent areas in either frozen or formalin-fixed tissues. Dual parameter flow cytometry, using an anti-pan-T lymphocyte antibody, revealed that greater than 99% of the cells stained by RA3.6B2 are a population distinct from T lymphocytes. F46A was shown to stain a sub-population of those cells stained with RA3.6B2. These antibodies may be useful in the identification of feline B lymphocytes using immunohistochemistry and flow cytometry and thereby provide additional tools to study B lymphocyte ontogeny and the significance of lymphocyte phenotype in lymphoid neoplasia in cats.     

快速诊断猪丹毒免疫组织化学方法开发和验证

本研究的目的,是研制一种能够快速检测猪红斑丹毒丝菌(Erysipelothrix rhusiopathiae)的免疫组织化学方法(Immunohistochemical,IHC).猪红斑丹毒丝菌血清1a、1b和2型与猪群临床疾病关系最为密切.试验所用血清1a、1b和2型的抗血清在兔体内制备,混合后用于福尔马林固定、石蜡包埋的猪组织(肺、心、脾脏和皮肤)切片.IHC检测结果与用来自实验性攻毒猪(6头抗生素治疗,8头未进行治疗)以及提交艾奥瓦州立大学兽医诊断实验室的田间病例(n=170)的样本直接细菌培养后所获结果进行比较,结果发现,直接细菌培养法的结果与IHC染色分析结果高度一致.本研究表明,IHC法用于检测福尔马林固定、石蜡包埋组织样本的猪红斑丹毒丝菌抗原有高度的灵敏性和特异性.由此表明,在遇到病猪在提交诊断实验室进行诊断前已用抗生素进行了治疗、且组织器官经直接细菌培养结果为阴性的情况下,用IHC法进行诊断尤其有用.此外,研究表明,IHC法对检测皮肤病变中的猪红斑丹毒丝菌抗原特别实用,而这些病变在进行细菌培养时往往显示为阴性.     

Application of anti-BCG antibody for rapid immunohistochemical detection of bacteria, fungi and protozoa in formalin-fixed paraffin-embedded tissue samples

The applicability of an anti-Mycobacterium bovis (BCG) antibody-based immunohistochemistry (IHC) procedure was investigated using everyday veterinary pathological samples collected from 13 different animal species. Fifty-one formalin-fixed and paraffin-embedded tissue samples were selected for this study. Forty, 4 and 7 tissue samples contained different species of bacteria, fungi and protozoa, respectively. Three serial sections were prepared in each case. Two sections were pre-treated with enzyme and heat, respectively, while the last section was not pre-treated. In seven cases the sensitivity of histochemical staining (HSM), IHC and bacteriological culture were compared. Heating of the sections in a microwave oven was the most effective method in the case of almost all pathogens used. Strong or moderate positive reactions were observed for 26 bacterial species, all fungal and 2 protozoal species, while weak reactions occurred for 2 bacterial and 1 protozoal species. Only 4 protozoal and 12 bacterial species, including Leptospira and all the five Mycoplasma species examined, showed no reaction in this test. IHC had almost the same sensitivity as bacteriological culture and was more sensitive than HSM. The IHC method presented here should be preferred to HSM as a general screening tool in cases where pathological lesions suspicious for infections are evident and no microorganism can be cultured in vitro or only formalin-fixed tissue samples are available for the laboratory examination.     

Identification of Toxoplasma gondii and Encephalitozoon cuniculi by immunoperoxidase techniques and electron microscopy, in stored, formalin-fixed, paraffin-embedded tissue

The peroxidase-anti-peroxidase (PAP), avidin-biotin-complex (ABC) techniques, and electron microscopy, were used to identify protozoan parasites in formalin-fixed material from routine necropsy cases. The material comprised paraffin blocks, that had been stored for up to 18 years, from 18 cases of suspected toxoplasmosis and encephalitozoonosis. With the ABC method a higher dilution of primary antibody could be used than with the PAP method. However, with both methods, a distinct reaction occurred with appropriate dilutions. The age of the material did not seem to effect the result. Frequently, phagocytized and necrotic parasites were also stained. Gystozoites (bradyzoites) of T. gondii were stained faintly compared to the endozoites (tachyzoites). A Toxoplasma-like parasite from a dog did not react with anti-Toxoplasma serum, and ultrastructurally it proved to be consistent with an unidentified cyst-forming sporozoan parasite previously reported in dogs. Electron microscopy based on paraffin-embedded tissue, seems to be a valuable method for identification of protozoan parasites, and thus provide a supplement or alternative to the immunoperoxidase methods.     

Identification of porcine macrophages with monoclonal antibodies in formalin-fixed,paraffin-embedded tissues

Here we report the successful use of monoclonal antibodies (mAbs) BL1H7, BA1C11 (anti-SWC3) and 4E9 for immunohistochemical identification of macrophages in formalin-fixed, paraffin-embedded porcine tissues. Retrieval of antigen reactivity was achieved by heating the slides in a domestic pressure cooker, which makes the technique suitable for the routine laboratory. This method allows to perform retrospective studies in routinely processed tissues and may be useful to investigate the role of macrophages in different pathological processes.     

Histological and genotypical characterization of feline cutaneous mycobacteriosis: a retrospective study of formalin-fixed paraffin-embedded tissues

Twenty-nine cases presumptively diagnosed as feline cutaneous mycobacteriosis were evaluated microscopically with haematoxylin and eosin and modified Fite's stained sections using archived formalin-fixed paraffin-embedded tissue specimens. Lesions were characterized histologically as feline leprosy (7 cases lepromatous and 16 cases tuberculoid) or atypical mycobacteriosis (3 cases); three cases did not fit these criteria and were classified as 'miscellaneous'. Actinomycetales-specific polymerase chain reaction (PCR) of variable regions 1, 2 and 3 of the 16S ribosomal RNA (rRNA) gene and subsequent sequence analysis of the amplicons were performed to identify the species of mycobacteria associated with each case. Together, this study identified 10 different Actinomycetales organisms with greater than 98% nucleotide sequence identity to named species, nine were of the genus Mycobacterium and eight were associated with feline leprosy (both lepromatous and tuberculoid). Based on this study, we conclude that feline cutaneous mycobacteriosis should be considered as a syndrome with varied clinical and histological presentations associated with a variety of different Mycobacterium species, organisms other than Mycobacterium sp. may be associated with feline cutaneous mycobacteriosis lesions, and molecular diagnostic techniques can be an important tool for identifying agents associated with lesions of feline cutaneous mycobacteriosis.     

Immunohistochemical identification of B and T lymphocytes in formalin-fixed, paraffin-embedded feline lymphosarcomas: relation to feline leukemia virus status, tumor site, and patient age.

The lymphocyte phenotype of 70 formalin-fixed, paraffin-embedded feline lymphosarcomas (LSAs) was determined immunohistochemically using a T cell polyclonal antibody, and a B cell monoclonal antibody. Forty-seven of 70 (67%) tumors were T cell, 19/70 (27%) were B cell, and 4/70 (6%) did not stain with either marker. Thirty-eight of 70 (54%) tumors were positive for feline leukemia virus (FeLV) antigen by immunohistochemistry (IHC), and 52/70 (74%) tumors were positive for FeLV DNA using the polymerase chain reaction (PCR). B cell tumors were as frequently FeLV-positive as T cell tumors using either IHC or PCR. Intestinal tumors were more likely to be B cell than T. The incidence of B and T cell tumors was not different among young (< or = 3 y), middle-aged (> 3 y to < or = 8 y), and old (> 8 y) cats. Both B and T cell tumors from old cats were FeLV-positive more often by PCR than by IHC. Feline leukemia virus DNA but not antigen, was detected in B cell tumors and intestinal tumors from cats > 8 y as often as it was detected in B cell tumors and intestinal tumors from cats < or = 8 y. Previously, most B cell and intestinal tumors from old cats were considered to be negative for FeLV. Here, the results suggest involvement of latent or replication-defective forms of the virus in such tumors from old cats. This study supports a role for FeLV in feline B cell as well as T cell tumorigenesis.     

Helicobacter spp. in the saliva,stomach, duodenum and faeces of colony dogs

The role of Helicobacter spp. infection in canine gastrointestinal disease is unclear and routes of transmission are of epidemiological and zoonotic importance. The aim of this study was to identify Helicobacter spp. in the saliva, stomach, duodenum and faeces of dogs using a multiplex PCR, and to evaluate any attendant histopathological changes. Helicobacter canis was the most common species detected in saliva and faeces and no correlation between the presence of Helicobacter spp. and histopathological changes in either the stomach or duodenum was observed. All dogs examined were co-infected with up to four species of the organism. This is the first time these bacteria have been studied at species level at multiple sites within the canine alimentary tract.     

Isolation and detection of Fasciola hepatica DNA in Lymnaea viatrix from formalin-fixed and paraffin-embedded tissues through multiplex-PCR

Detection of Fasciola hepatica infection in Lymnaea viatrix through analysis of histological cuts is based upon morphological characters of the parasite during the intra-mollusk phase of parasitism. At this stage, trematode forms are very similar and, thus, very difficult to differentiate. Specific detection may also be impaired by the presence of other helminthes in the mollusk. Histological samples are usually fixed in formalin, embedded in paraffin, sectioned and HE stained. In the current study, a method for the extraction of DNA from formalin-fixed, paraffin-embedded tissues was standardized by means of deparaffinizing with xylol and digesting with proteinase K. Extracted DNA was amplified in a multiplex-PCR, by using simultaneous primers in a single reaction under high stringency conditions. Results showed specific amplification of DNA from the trematode and the snails. The technique was sensitive enough to detect F. hepatica infections in L. viatrix, in histological sections in which the presence of larval stages could not be observed through brightfield microscopy. The profiles generated were: stair bands referring to F. hepatica DNAmt amplification; a band of 1200 bp referring to L. viatrix ITS and another of 1300 bp referring to F. hepatica ITS and other trematodes. Multiplex-PCR has shown to be a fast, safe, highly sensitive and specific method, which is able to amplify DNA from fixed tissues, despite a low DNA quantity and its degradation caused by fixation processes. Such methodology may be useful in studies on fascioliasis epidemiology, enabling the use of material from histological collections.     

PCR detection of Clostridium chauvoei in pure cultures and in formalin-fixed,paraffin-embedded tissues

The polymerase chain reaction (PCR) was used to amplify specific segments of the 16S ribosomal RNA gene of Clostridium chauvoei, a major pathogen of ruminants. Three sets of primers were used to produce amplicons of 159, 836 and 959 base pairs (bp), respectively. The PCR was evaluated by testing clinically important strains of Clostridium, including 21 strains of C. chauvoei, five strains each of Clostridium septicum and Clostridium perfringens and two strains each of Clostridium novyi, Clostridium histolyticum and Clostridium sordellii. Both purified DNA and biomass from pure cultures of each of these microorganisms were evaluated as templates in the PCR. In addition, extracts of formalin-fixed, paraffin-embedded tissues of eight sheep experimentally inoculated with C. chauvoei or C. septicum (four animals each) were also tested by the PCR using the three sets of primers. Purified DNA template of all C. chauvoei strains produced PCR amplicons of the expected size for all three primer pairs. However, when biomass from pure cultures of C. chauvoei or tissue extracts were used as templates, only the primer pair designed to produce the 159bp amplicon gave consistently positive results. No positive results were obtained with any primer pair when purified DNA or biomass from pure cultures of non-target clostridial species were used as templates. Therefore, the PCR primer sets appear to be very specific for identifying C. chauvoei in both cultures and tissues.     

Detection of Streptococcus suis by in situ hybridization, indirect immunofluorescence, and peroxidase-antiperoxidase assays in formalin-fixed, paraffin-embedded tissue sections from pigs.

Streptococcus suis is an important pathogen in pigs and is considered a zoonotic agent. To aid diagnosis of infection caused by S. suis, a species-specific probe targeting 16S ribosomal RNA was designed and used for fluorescent in situ hybridization. Two additional immunohistochemical detection methods, an indirect immunofluorescence assay and a peroxidase-antiperoxidase method, using polyclonal antibodies also were developed. The specificity of the oligonucleotide probe was examined by whole-cell and dot-blot hybridization against reference strains of the 35 serotypes of S. suis and other closely related streptococci and other bacteria commonly isolated from pigs. The probe was specific for S. suis serotypes 1-31. The specificity of the polyclonal antibodies, which has previously been evaluated for use in diagnostic bacteriology for typing of serotype 2, was further evaluated in experimentally infected murine tissue with pure culture of different serotypes of S. suis, related streptococci, and other bacteria commonly found in pigs. The polyclonal antibodies against S. suis serotype 2 cross-reacted with serotypes 1 and 1/2 in these assays. The in situ hybridization and the immunohistochemical methods were used for detection of S. suis in formalin-fixed, paraffin-embedded tissue sections of brain, endocardium, and lung from pigs infected with S. suis. The methods developed were able to detect single cells of S. suis in situ in the respective samples, whereas no signal was observed from control tissue sections that contained organisms other than S. suis. These techniques are suitable for determining the in vivo localization of S. suis for research and diagnostic purposes.     

Development of a polyclonal-antibody-based immunohistochemical method for the detection of type 2 porcine circovirus in formalin-fixed, paraffin-embedded tissue.

An immunohistochemical method for the detection of type 2 porcine circovirus (PCV2) in paraffin-embedded tissue was developed. Rabbits were inoculated with purified PCV2 to obtain a polyclonal antiserum. Antiserum was applied to sections of porcine tissue that contained lesions consistent with postweaning multisystemic wasting syndrome and in which PCV2 genome had been demonstrated by in situ hybridization. In all cases (18/18), the density and distribution of positive cells detected by in situ hybridization or immunohistochemistry were identical. The immunohistochemical method is more rapid and less expensive than in situ hybridization and is thus more suitable for routine diagnostic use.     

Feline leukemia virus detection by immunohistochemistry and polymerase chain reaction in formalin-fixed, paraffin-embedded tumor tissue from cats with lymphosarcoma.

The prevalence of feline leukemia virus (FeLV) antigen and DNA was assessed in formalin-fixed, paraffin-embedded tumor tissues from 70 cats with lymphosarcoma (LSA). Tissue sections were tested for FeLV gp70 antigen using avidinbiotin complex (ABC) immunohistochemistry (IHC); DNA was extracted and purified from the same tissue blocks for polymerase chain reaction (PCR) amplification of a 166 base pair region of the FeLV long terminal repeat (LTR). Results were related to antemortem FeLV enzyme-linked immunosorbent assay (ELISA) for serum p27 antigen, anatomic site of LSA, and patient age. Viral DNA was detected by PCR in 80% of cases and viral antigen by IHC in 57% of cases. Seventeen cases were PCR-positive and IHC-negative; one case was PCR-negative and IHC-positive. Clinical records included FeLV ELISA results for 30 of 70 cats. All 19 ELISA-positive cats were positive by PCR and IHC; of the 11 ELISA-negative cats that were negative by IHC, seven were positive by PCR. When evaluated according to anatomic site, FeLV DNA and antigen were detected less frequently in intestinal LSAs than in multicentric and mediastinal tumors. Lymphosarcoma tissues from cats < 7 yr were several fold more likely to be positive for FeLV antigen by IHC than were tumors from cats > or = 7 yr. However, there was no significant difference in PCR detection of FeLV provirus between LSAs from cats < 7 yr and those > or = 7 yr.(ABSTRACT TRUNCATED AT 250 WORDS)     

新生仔猪肝脏胰腺和胃发育的初步研究

试验研究了1～7日龄仔猪肝脏、胰腺和胃的生长发育。结果表明,自然哺乳仔猪3日龄的肝脏、胰腺和胃的绝对重量分别较初生时增加了94.09%P<0.01、101.53%P<0.01和74.25%P<0.01,而同期体重才增加了24.3%P<0.05,而4～7日龄其绝对重量的增加有所降低,分别为64.86%P<0.01、52.75%P<0.01和55.85%P<0.01,同期体重增加了63.8%P<0.01因此,1～3日龄肝、胰腺和胃的发育较4～7日龄的迅速。     

The content of fresh material, DNA and RNA in different tissues of turkeys during growth. 2. Analysis of the myocardium, the liver, the lungs, the kidney, the pancreas, the spleen and the duodenum]

The highest DNA concentration so far recorded from organs of turkey chicken one day after hatching was the lung value of 8.61 +/- 0.20 mg/g of fresh matter, while the lowest level amounted to 1.92 +/- 0.11 mg/g and was linked to the myocardium. DNA concentrations in the spleen went up from 5.93 +/- 0.26 mg/g on the first day to 13.04 +/- 0.93 mg/g on the 224th day. The highest total amount of DNA on the 224th day was 314.6 +/- 30.23 mg/g in the liver. A high RNA concentration on the first day from hatching was 8.74 +/- 0.14 mg/g in the liver and the lowest 2.51 +/- 0.12 mg/g in the myocardium. DNA levels for the whole period under review (first through 224th days) rose by the following factors: 246.3 in the spleen, 87.4 in the pancreas, 76.6 in the liver, 60 in the duodenum, 40.5 in the kidneys, 18 in the lung, and 31.3 in the myocardium. Functional implications relating to DNA, RNA, and protein variations are discussed in some detail.     

Immunohistochemical detection of Mycoplasma agalactiae in formalin-fixed,paraffin-embedded tissues from naturally and experimentally infected goats

Samples from the mammary tissue of 14 lactating goats (12 naturally infected and two experimentally infected) were examined for the presence of Mycoplasma agalactiae. A monoclonal antibody (5G12) was applied to formalin-fixed, paraffin-wax-embedded sections and labelled by the avidin-biotin peroxidase complex (ABC) method. Histological examination of tissue sections revealed strong immunoreactivity in all animals included in the study. Mycoplasma agalactiae antigen was mainly detected in the cellular debris at the periphery of purulent exudates present within lactiferous sinuses, and lactiferous and interlobular ducts. In addition, M. agalactiae organisms appeared in the cytoplasm of the epithelium of ducts, and in infiltrating macrophages and neutrophils within the ducts, alveoli, interstitial tissue and regional lymph node sinuses. It is concluded that this monoclonal antibody-based immunohistochemical technique is an efficient and specific method for the post-mortem detection of M. agalactiae in cases of clinical mastitis as well as being a useful tool for the study of the route of infection and cellular types involved during mastitis caused by this organism.