PCR技术检测饲料中沙门氏菌的应用研究

用聚合酶链反应(PCR)技术检测饲料中沙门氏菌,对已知被沙门氏菌污染的动物饲料培养物均能检出阳性,说明本方法对检测饲料中沙门氏菌具有特异性高、灵敏、快速等特点,适用于快速、准确地检测饲料中沙门氏菌的需要。     

Policies for the vaccination of cats and dogs in New Zealand veterinary practices

AIMS: To determine current practices and attitudes towards vaccination of dogs and cats of veterinarians in New Zealand; the methods used for informing clients on which vaccines to use, and the preferred site for vaccination of cats.

METHODS: A postal questionnaire was sent to all 483 listed veterinary practices in New Zealand during February 2012. Some questions were specific to pet dogs, cats, or working farm dogs. Responses were categorised according to practice type and geographical region of the respondent. Factors associated with respondent recommendation of annual vaccination with modified live viral (MLV) vaccines were examined using logistic regression analysis. Vaccines that were considered to be essential for every animal were defined as core; those that may be recommended for animals whose location or lifestyle placed them at risk, were defined as non-core.

RESULTS: There were 204 useable returns, equivalent to a response rate of 42.2%, distributed across the country. Annual vaccination with MLV vaccines of dogs was recommended by 54/198 (27.3%) respondents, and of cats by 107/181 (59.1%) respondents. Factors associated with the recommendation of annual administration of MLV vaccines to dogs included being a companion animal practice, a desire for policies on vaccination to be left to individual clinics, and having one veterinarian in the practice. Administration of the final vaccination for puppies was recommended at ≥14 weeks old by 55/185 (29.7%) respondents, and for kittens at ≥13 weeks old by 42/183 (23%) respondents. Of respondents that administered MLV vaccines annually, 62/103 (60.2%) believed reducing the frequency of vaccination would reduce income, and 52/103 (50.5%) considered it would have a negative effect on animal health. Advice to enable clients to decide which non-core vaccines were administered was given by 181/199 (91%) respondents. Factors considered when recommending a vaccine included consideration of risk to individual patients (190/203; 93.6%), requirements of boarding kennels/catteries (165/203; 81.3%) and clinic vaccination policy (142/203; 70%). The preferred site for administering MLV vaccines to cats was the dorsal neck or inter-scapular region (137/198; 69.2%). Amongst respondents, 18 wanted disease surveillance information to allow for truly informed decisions to be made about vaccination.

CONCLUSIONS AND CLINICAL RELEVANCE: Veterinarians can now compare their own vaccination practices and attitudes with those of veterinarians nationally, and internationally. There is a need for national surveillance information and for continued education of the public and commercial kennel and cattery owners for optimal vaccination strategies to be developed.     

Digestibility for dogs and cats of meat and bone meal processed at two different temperature and pressure levels*

This study evaluated the effect of two rendering processes applied to meat and bone meal (MBM) production, associated or not with extrusion, by measuring diet apparent total tract digestibility of dogs and cats, and true digestibility of amino acids (AA) by cecectomized roosters. Four diets were evaluated, with the main protein source as follows: conventional and extruded MBM; high temperature and pressure (HPT; 135 °C, 3 bar, 20 min) and extruded MBM; conventional non‐extruded MBM; HPT non‐extruded MBM. Nutrient digestibility and food metabolizable energy content were evaluated with dogs and cats by the method of total collection of faeces. True AA digestibility was evaluated by a precision‐fed assay with cecectomized roosters. The evaluated MBM had high‐ash content. The HPT process of MBM increased the digestibility of crude protein of the diets by cats, and the true digestibility of several AA by cecectomized roosters. The extrusion process did not modify the apparent total tract nutrient digestibility of MBM by dogs and their amino acid digestibility by roosters, but increased the digestibility of the dietary protein by cats. Dogs fed HPT MBM presented a higher urea post‐prandial response, suggesting reductions in bioavailability and protein synthesis from absorbed AA.     

Use of nested polymerase chain reaction (PCR) for detection of retroviruses from formalin-fixed, paraffin-embedded uveal melanomas in cats

Thirty-six formalin-fixed, paraffin-embedded enucleated globes from cats with a diagnosis of diffuse anterior uveal melanoma were obtained. Sections of tumor were excised, deparaffinized, and subjected to nested polymerase chain reaction (PCR) to identify proviral DNA sequences from the feline leukemia virus (FeLV)–feline sarcoma virus (FeSV; 36 eyes), and the feline immunodeficiency virus (FIV; 18 eyes). All samples tested were negative for FIV DNA. Three samples were positive for FeLV–FeSV DNA. This is the first reported evidence of a possible link between naturally occurring feline anterior uveal melanoma and the presence of FeLV–FeSV DNA.     

李斯特氏菌因子血清的制备及食品检测的免疫磁性分离-PCR(MIPA)方法的建立

首先进行了李斯特氏菌因子血清的研制,制备出了可对所有李斯特氏菌分型的 １５ 个 Ｏ 抗原因子和４ 个 Ｈ 抗原因子的因子血清。利用复合因子血清的多克隆抗体包被磁性球,对食品中的单核细胞增多性李斯特氏菌进行免疫磁性分离,并与 Ｐ Ｃ Ｒ 方法相结合,建立了检测食品中单核细胞增多性李斯特氏菌的 Ｍ Ｉ Ｐ Ａ方法（免疫磁性分离—聚合酶链反应方法,ｍ ａｇｎ ｅｔｉｃ ｉｍ ｍ ｕｎｏｐｏｌｙｍ ｅｒａｓｅ ｃｈａｉｎ ｒｅａｃｔｉｏｎ ａｓｓａｙ）。对菌液、模拟样品的检测表明,本方法能够有效地克服食品基质、培养基成分和杂菌对 Ｐ Ｃ Ｒ 检验的干扰作用。食品样品在 Ｅ Ｂ增菌液中增菌 １２ ｈ 后,检测的敏感度达 ５ Ｃ Ｆ Ｕ／ｍ Ｌ,可以在 ２０ ｈ 内完成检测。本方法对实际食品样品的检测结果,与国家标准检验方法检测的结果一致。     

A multiplex quantitative real-time polymerase chain reaction panel for detecting neurologic pathogens in dogs with meningoencephalitis

Meningoencephalitis (ME) is a common inflammatory disorder of the central nervous system in dogs. Clinically, ME has both infectious and non-infectious causes. In the present study, a multiplex quantitative real-time polymerase chain reaction (mqPCR) panel was optimized for the detection of eight canine neurologic pathogens (Blastomyces dermatitidis, Cryptococcus spp., Neospora caninum, Borrelia burgdorferi, Bartonella spp., Toxoplasma gondii, Ehrlichia canis, and canine distemper virus [CDV]). The mqPCR panel was subsequently applied to 53 cerebrospinal fluid (CSF) samples collected from dogs with ME. The analytic sensitivity (i.e., limit of detection, expressed as molecules per 1 µL of recombinant vector) was 3.8 for CDV, 3.7 for Ehrlichia canis, 3.7 for Bartonella spp., 3.8 for Borrelia burgdorferi, 3.7 for Blastomyces dermatitidis, 3.7 for Cryptococcus spp., 38 for Neospora caninum, and 3.7 for Toxoplasma gondii. Among the tested CSF samples, seven (15%) were positive for the following pathogens in decreasing order of frequency: Cryptococcus spp. (3/7), Blastomyces dermatitidis (2/7), and Borrelia burgdorferi (2/7). In summary, use of an mqPCR panel with high analytic sensitivity as an initial screen for infectious agents in dogs with ME could facilitate the selection of early treatment strategies and improve outcomes.     

Application of the polymerase chain reaction (PCR) in veterinary diagnostic virology

The polymerase chain reaction has become an important diagnostic tool for the veterinary virologist. Conventional methods for detecting viral diseases can be laborious or ineffective. In many cases PCR can provide a rapid and accurate test. In this article we explain the basic principles of PCR and supply a reference list of its uses in diagnostic veterinary virology.Abbreviations BLV bovine leukaemia virus - BVDV bovine viral diarrhoea virus - DNA deoxyribonucleic acid - ds double-stranded - ELISA enzyme-linked immunosorbent assay - PCR polymerase chain reaction - RNA ribonucleic acid - ss single-stranded - TK thymidine kinase     

Detection of Ehrlichia platys in dogs in Australia

OBJECTIVE: To describe the detection of Ehrlichia platys in free-roaming dogs in Central Australia. PROCEDURE: Blood samples were collected from four dogs and examined for bacterial 16S ribosomal DNA using Polymerase Chain Reaction (PCR)-based assays. The three positive samples obtained were then sequenced and identification of the PCR product carried out. As a result of all three samples being identical to or closely related to part of the 16S rRNA gene of E. platys, blood samples were subsequently obtained from a further 24 dogs. These samples were screened using a PCR-assay to determine the presence of Ehrlichia DNA using genus-specific primers. The positive samples obtained from the screening process were then subjected to a further PCR-assay using E. platys specific primers. RESULTS: Of 28 dogs sampled, Ehrlichia DNA was detected in the blood of 13 dogs. Sequencing of the amplicons obtained indicated a high homology with the 16S rRNA gene for E. platys. When the E. platys-specific PCR was performed for 10 of those dogs, the 678 bp product obtained from the PCR amplification confirmed the identification as part of the 16S rRNA gene of E. platys in all 10 dogs. CONCLUSION: This study reports for the first time Ehrlichia carriage by dogs in Australia. It also indicates the usefulness of the PCR technique in rapidly and accurately identifying diseases that are otherwise difficult to detect. By using universal primers directed against bacterial 16S ribosomal DNA and sequencing analysis, the detection of potentially pathogenic Ehrlichia organisms that had not previously been found in Australia has been made possible.     

Identification of parvovirus in the bone marrow of eight cats

Objective To determine if canine parvovirus (CPV) or feline panleucopenia virus (FPV) genomic sequences are present in adult feline bone marrow samples. Design Bone marrow samples were obtained from 32 semi‐feral cats that were euthanased at an animal shelter. DNA was extracted and subjected to conventional polymerase chain reaction (PCR) designed to determine if CPV or FPV DNA was present. Positive PCR products were purified, cloned and sequenced to differentiate between CPV and FPV. Results Eight of the bone marrow samples contained parvoviral DNA (7 CPV, 1 FPV). Conclusion CPV and FPV DNA can be found in the bone marrow of healthy adult cats.     

Prevalence of bovine herpesvirus-4 infection in cats in Central Michigan

The gammaherpesvirus bovine herpesvirus-4 (BHV-4) has been isolated from a wide variety of animals, including lions and domestic cats. Although BHV-4 antibodies have been detected in normal cats and cats with urinary disorders, the epidemiology and pathogenic role of BHV-4 in cats is unknown. The purpose of this study was to determine the prevalence of BHV-4 antibodies and viral nucleic acid in a population of free-roaming cats. Plasma and peripheral blood leukocyte samples were collected from 52 male and 52 female free-roaming cats impounded at a regional animal control facility in Central Michigan. Plasma concentrations of BHV-4 antibodies were measured with an indirect fluorescent antibody test. Peripheral blood leukocyte DNA was isolated, and a 2-stage polymerase chain reaction with heminested primers delineating a conserved portion of the BHV-4 glycoprotein B gene homologue was used to amplify BHV-4-specific DNA sequences. BHV-4 antibodies were detected in 38 (73%) male and 23 (44%) female cats. Seropositive cats were significantly more likely to be male than female (odds ratio = 3.22; P = .007). Cell-associated viremia was detected in 17 (33%) male and 11 (21%) female cats. Of the 61 seropositive cats, 23 (38%) had a detectable viremia; only 5 (12%) seronegative cats had detectable viremia. Seropositive cats were significantly more likely to be viremic than seronegative cats (OR = 4.30: P = .009). Our results suggest that BHV-4 infection may be more widespread in certain cat populations than previously reported. Furthermore, many cats seropositive for BHV-4 antibodies have a concurrent cell-associated viremia.     

应用PCR技术检测鹅细小病毒

根据鹅细小病毒GPVH1株结构蛋白VP1与VP3编码基因非重叠核苷酸序列设计了一对引物GPR1 /GPR2 ,用这对引物对感染器官内的GPR和接种鹅胚增殖的GPV进行PCR扩增 ,其结果引物GRP1 /GPR2能特异性地扩增GPVVP1 VP3区段核苷酸序列 ,扩增出与设计核苷酸片段大小相符的 0 6kb的序列 ,而对照的鹅副粘病毒cDNA及鸭瘟病毒 (DPR)DNA对照组均出现阴性结果。     

Detection of neoplastic lymphocytes in peripheral blood of dogs with lymphoma by polymerase chain reaction for antigen receptor gene rearrangement

BACKGROUND: Uniquely rearranged immunoglobulin and T-cell receptor gene sequences can be amplified and electrophoretically separated by size to detect a clonal population of lymphocytes. OBJECTIVE: The purpose of this study was to determine whether the polymerase chain reaction (PCR) detects neoplastic (clonal) lymphocytes more frequently than do microscopic methods. METHODS: We identified neoplastic lymphocytes in peripheral blood by both routine and standardized microscopic examination of blood smears and by PCR amplification of blood-derived DNA and compared the 3 methods for frequency of detection of leukemic involvement. For standardized microscopic examination (200 leukocytes counted on Wright-Giemsa-stained blood smears), samples were categorized as negative (1% prolymphocytes, no lymphoblasts), or positive (>/=1 lymphoblast). A PCR-amplified sample was positive if 1 or 2 discrete bands were seen on the gel, or negative if no bands, a smear, or a faint ladder was seen. RESULTS: Using PCR, neoplastic lymphocytes were detected in peripheral blood 2.5 times more frequently than with routine or standardized microscopic evaluation. Eighty-three percent of samples negative by microscopy were positive by PCR. CONCLUSION: PCR is more sensitive than microscopy for the detection of clonal lymphocytes in peripheral blood. The results of this study also suggest that neoplastic lymphocytes circulate in peripheral blood at a higher frequency than previously reported. PCR may be useful for detecting or phenotyping lymphoma, monitoring response to therapy, identifying recurrence, and screening breeds at risk.     

Bartonella spp. DNA in cardiac tissues from dogs in Colorado and Wyoming

Background: Several Bartonella species (spp.) have been identified in dogs diagnosed with infectious endocarditis (IE) or myocarditis. Objective: To interrogate cardiac tissues of dogs with suspected IE for the presence of Bartonella spp. DNA of dogs in the Rocky Mountain states. Animals: Nine dogs with a clinical diagnosis of endocarditis from January 1990 to June 2008 were included. Methods: In this retrospective study, medical records at the Veterinary Teaching Hospital were searched. Animals were excluded if there was no diagnosis of IE in the original necropsy report. Paraffin embedded tissue blocks and medical records were available from 9 dogs. Total DNA was extracted from the cardiac tissues and assessed for Bartonella spp. DNA by 3 polymerase chain reaction (PCR) methods. For positive samples, the Bartonella spp. were determined by genetic sequencing or fluorogenic real‐time PCR. Results: Bartonella henselae DNA was amplified from the tissues of 7 dogs; Bartonella vinsonii subsp berkhoffii DNA was amplified concurrently from 3 dogs. Six dogs were from Colorado and 1 was from Wyoming. Flea or tick infestations were reported in 2 dogs. Conclusions and Clinical Importance: Bartonella spp. should be on the differential list for dogs in the Rocky Mountain states. The results emphasize the need for routine use of external parasite control products even in regions perceived to have low risk for flea and tick infestations.     

Relationship between retinoic acid receptor alpha gene expression and growth-inhibitory effect of all-trans retinoic acid on canine tumor cells

Retinoids show antitumor effects on human acute promyelocytic leukemia and other tumors via retinoid receptors. In dogs, the role of retinoid receptors in inhibiting tumor development remains unclear. To evaluate the correlation between the degree of expression of retinoic acid receptor alpha (RARalpha) mRNA and the antiproliferative effects of all-trans retinoic acid (ATRA) treatments, expression analysis of RARalpha mRNA and cell growth inhibition assay were performed on 17 established canine tumor cell lines, including 6 mammary gland tumor (MGT) cell lines, 3 osteosarcoma cell lines, 5 melanoma cell lines, and 3 mast cell tumor (MCT) cell lines. Among the cell lines investigated, all 3 MCT cell lines showed high expression of RARalpha, and the most effective cell growth inhibition was observed in ATRA-treated MCT cell lines. However, remarkable antiproliferative effects of ATRA treatments were not observed on other tumor cell lines with moderate or low RARalpha mRNA expression. As a result of the relationship between RARalpha mRNA expression and ATRA treatment with regression analysis, statistically significant correlation was suggested. Furthermore, real-time quantitative polymerase chain reaction analysis of RARalpha was performed on MCT tissue samples of dogs with spontaneous disease, and 5 of 9 tissues showed high expression. These results suggest that ATRA may be an effective antitumor agent for MCT in dogs, and that prior measurement of expression of RARalpha mRNA may be a good indicator of the effectiveness of ATRA treatment.     

Characterisation of lymphosarcomas in Australian cats using polymerase chain reaction and immunohistochemical examination

Objective To examine tumour tissue of cats with lymphosarcoma for the presence of feline leukaemia virus and feline immunodeficiency virus and analyse the immunophenotype of the tumours.
Design A retrospective study of feline lymphosarcoma cases.
Methods Formalin-fixed, paraffin-embedded tumour tissue of 14 feline lymphosarcomas was examined for the presence of feline leukaemia virus and feline immunodeficiency virus by polymerase chain reaction and immunohistochemistry. Using polyclonal and monoclonal antibodies against T and B lymphocytes, the phenotypic expression of the tumours was characterised.
Results No feline leukaemia virus antigen or proviral sequences were detected. Feline immunodeficiency virus proviral sequences were detected in two cases by polymerase chain reaction. Immunophenotyping of all 14 cases resulted in seven cases being classified as B-cell phenotype, four as T-cell phenotype, and the remaining three undetermined.
Conclusions In contrast to previous reports overseas, our results suggest that feline leukaemia virus infection appears to be an infrequent cause of lymphosarcoma in the cats that were necropsied. Feline immunodeficiency virus may have a role in lymphomagenesis. The potential role of feline immunodeficiency virus needs to be explored in more depth. Compared with most previous reports, B-cell tumours were more common than T-cell tumours in this series of cats.     

Determination of free and total cyst(e)ine in plasma of dogs and cats

BACKGROUND: In human blood, the amino acid cysteine forms disulfide bonds with itself and with other sulfhydryl compounds in their free form and with sulfhydryls in protein. Protein-bound cysteine is lost when plasma proteins are removed before amino acid analysis. OBJECTIVE: The purpose of this study was to assess the time course and extent of cyst(e)ine (cysteine + half-cystine) loss in dog and cat plasma. METHODS: An equal volume of 6% sulfosalicylic acid was added to plasma aliquots at 0, 2, 4, 10, 16, 24, 36, 48, 60, and 72 hours after separation of blood cells. Tris-2-carboxyethyl-phosphine hydrochloride (TCEP - HCl), a reducing agent, was used to regenerate total plasma cyst(e)ine after 3 months of sample storage (-20 degrees C). RESULTS: Initial free cyst(e)ine concentrations (mean +/- SEM) were higher in canine plasma (77 +/- 4 micromol/L) than in feline plasma (37 +/- 3 micromol/L). Free plasma cyst(e)ine concentrations in dogs and cats decreased after first-order kinetics, with a half-life of 23 and 69 hours, respectively. Total plasma cysteine after TCEP - HCl treatment was similar for dogs (290 micromol/L) and cats (296 micromol/L), but the percentage of free cysteine was higher (P = .02) in dogs (27%) than in cats (13%). Over half of the cyst(e)ine, homocysteine, cysteinylglycine, and glutathione were bound in vivo to plasma proteins. CONCLUSION: These results emphasize the importance of removing plasma proteins within 1 hour after blood collection for reliable assay of free plasma cyst(e)ine.