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1.
XX/XY chimerism and freemartinism in a female llama co-twin to a male.   总被引：2，自引：0，他引：2  
A 20-month-old female llama was examined because at the time of mating, the male llama was apparently unable to achieve intromission. The female llama had been born co-twin to a male. On physical examination, the vaginal vestibule appeared to end in a blind pouch, and the uterus, cervix, and ovaries could not be identified during transrectal palpation or ultrasonography. Karyotyping was done, and 43% of blood lymphocytes had 2 X chromosomes, and 57% had 1 X and 1 Y chromosome. All skin fibroblasts had 2 X chromosomes. A diagnosis of freemartinism and XX/XY chimerism was made. Because conception of twins may be more common in llamas than birth of twins, it is possible that freemartinism could develop in singleton females, if, for instance, a male twin was conceived and died after the placentas had anastomosed. More widespread use of karyotyping in llamas with congenital defects of the reproductive tract will help to define the incidence of freemartinism.  相似文献   

2.
OBJECTIVE: To evaluate and implement rapid molecular diagnostic techniques for the detection of foot and mouth disease virus (FMDV) suitable for use in Australia. DESIGN: Two PCR TaqMan assays targeted to the FMDV internal ribosome entry site or the 3D polymerase coding region for the rapid detection of FMDV were evaluated using non-infectious materials to determine the test most appropriate for implementation as part of Australia's national preparedness for the rapid detection and diagnosis of FMD outbreaks. RESULTS: Two published tests (PCR TaqMan assays targeted to the FMDV IRES region or the FMDV 3D polymerase coding region) were evaluated for their ability to detect FMDV genetic material in non-infectious FMDV ELISA antigen stocks held at Australian Animal Health Laboratory. Both tests were able to detect FMDV genetic material from strains O1 Manisa, O-3039, A22, A24, A Malaysia, C, Asia 1 and SAT 1, 2 and 3. With the exception of Asia 1, the TaqMan assay targeted to the FMD 3D polymerase coding region had Ct values equal to or lower than for the TaqMan assay targeted to the IRES region suggesting that this test may provide broader serotype detection and sensitivity. However, the TaqMan assay directed to the FMDV IRES is the only one to date to have undergone substantial evaluation using clinical samples collected during an outbreak. The greatest differences observed were for O-3039, SAT 1, and 3. CONCLUSION: Given the ease of setting up both tests, AAHL currently runs both tests on highly suspect FMD investigations to provide independent confirmation of the absence of FMDV because the tests are focused on two independent regions of the FMDV genome. These tests add substantially to Australia's preparedness for FMD diagnosis complementing the already well-established virus isolation and antigen capture ELISA tests for index case diagnosis of FMD in Australia.  相似文献   

3.
A concurrent infection of chickens with infectious laryngotracheitis virus (ILTV), a herpesvirus, and fowlpox virus (FWPV), an avipoxvirus, is described. Two techniques, an immunohistochemistry (IHC) technique and a multiplex polymerase chain reaction (PCR), were used to examine 11 tissue samples from chickens clinically diagnosed as FWPV-infected, but only IHC was used to examine six tissue-paraffin blocks prepared from turkeys suspected of having FWPV infection. By multiplex PCR, both FWPV and ILTV were detected from three chicken samples (FI-90, FI-93, and FI-94); both FWPV and ILTV were detected from only two samples (FI-93 and FI-94) by IHC. All chicken samples were positive for FWPV by both PCR and IHC. Viral DNA from these samples was further confirmed by restriction enzyme analysis. When turkey samples were analyzed by the double-stain IHC, all six samples showed the presence of FWPV antigens, but no ILTV antigens. The double IHC technique, using monoclonal antibodies against FWPV and ILTV, was successful in simultaneous demonstration of specific FWPV and ILTV antigens colocalized in infected tissue samples as well as within individual cells. This paper emphasizes the importance of reliable tests that detect specifically the presence of ILTV and FWPV in infected tissue samples. The multiplex PCR assay holds potential to be versatile, rapid, and more sensitive (100%) than IHC (67%) for the simultaneous detection of two different avian viruses. Furthermore, the presence of mixed infection should always be kept in mind in the virologic analysis of respiratory sickness of poultry.  相似文献   

4.
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5.
Two hundred and thirty six semen samples were collected from 120 bulls, 60 buffalo-bulls, and 56 rams located on farms of known history of infection with Chlamydophila species. All semen samples were examined by polymerase chain reaction (PCR) and cell culture techniques for detection of Chlamydophila species. The primers were selected to allow the amplification of all target species in a single reaction by identifying conserved sequences in the omp2 gene. PCR assay detected more positive samples (36) from the semen samples collected from different animal species than were detected by the culture method (21). The results indicated that all culture-positive semen samples (21) from different species were PCR positive. The detection limit of the PCR assay was determined with DNA extracted from fourfold serial dilution of C. abortus (B577) and C. pecorum (11/88) cultures and found to be 0.25 inclusion-forming units (IFU) per PCR, while the culture method could not detect less than 4 IFU. This is the first report using PCR for the detection of Chlamydophila species in buffalo-bulls' semen and the assay provides a simple, sensitive, rapid, and reliable means for the detection and identification of the organism.  相似文献   

6.
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7.
A rapid polymerase chain reaction (PCR) assay was developed for detecting Salmonella in faeces of horses and assessed on samples from horses admitted to a veterinary hospital. Direct detection was achieved by amplification of part of ompC after extraction of DNA from faeces using a spin column method to reduce the amount of inhibitory substances in samples. An internal positive control was included to detect false negative results. While the sensitivity of the PCR assay was less than culture when assessed on faeces inoculated with Salmonella, its sensitivity on faecal samples obtained from horses was much greater than culture. Salmonella DNA was detected in 40% of faecal samples using the PCR assay while Salmonella were cultured from only 2% of the samples. The PCR assay has potential for use in either routine diagnosis or for detection of the carrier status in animals.  相似文献   

8.
A real-time PCR assay based on TaqMan probe chemistry was developed for the detection of Theileria parva DNA in blood samples. It uses a Theileria genus-specific PCR primer set and a T. parva-specific probe to amplify and hybridize with a species-specific part of the 18S rRNA gene of the parasite. The test was evaluated using positive and negative reference blood samples and shown to be specific for T. parva. Analytical sensitivity was determined by testing a dilution series of T. parva positive blood. It was shown to be able to detect parasitaemia as low as 2 × 10(-6)%. The Taqman assay results were also compared with that obtained with the real-time hybridization probe PCR assay, which is currently employed as the official test for the diagnosis of T. parva infections in buffalo and cattle and was shown to be equally sensitive. A panel of 1164 field samples was screened using both assays and 164 samples tested positive in both tests, indicating a good correlation.  相似文献   

9.
OBJECTIVE: To develop a multiplex polymerase chain reaction (PCR) assay for the rapid detection of Brucella ovis, Actinobacillus seminis, Histophilus somni in fresh ram semen samples. DESIGN: The multiplex assay was based on the single PCR assays published for the detection of A seminis and B ovis, and the forward primer published for the detection of H somni; an alternative reverse primer for H somni was designed in this study. PROCEDURE: Culture and PCR of 295 fresh semen samples were carried out. RESULTS: The multiplex PCR was far more successful in the detection of H somni (45/295) than culture (23/295). A seminis was also detected in more semen samples by multiplex PCR (29/295) than culture (13/295) and B ovis was detected in three samples using both PCR and culture. No amplifications were detected with DNA from a range of bacterial isolates including species associated with epididymitis in rams. CONCLUSION: This PCR could be used as a complementary test, or alternative to culture of ram semen and other biological samples for the detection B ovis, H somni and A seminis.  相似文献   

10.
荧光定量RT-PCR检测禽白血病病毒方法的建立及应用   总被引：5，自引：0，他引：5  
gag基因是禽白血病病毒(ALV)的群特异性基因,根据GenBank中登录的gag基因的保守序列(E46390),设计合成了1对引物,建立了基于SYBR GreenI模式的检测禽白血病病毒的实时荧光定量RT-PCR方法。该方法只对目的基因有扩增,对其它无关病毒核酸无特异性扩增;扩增效率为94.6%;在8.2×102～8.2×107拷贝的范围内有很好的线性关系(Y=3.46X+31.8,r=0.9995);最低可检测82个拷贝的病毒核酸,与普通PCR方法相比,灵敏度高出1000倍。组内CV为0.28%～1.45%;组间CV为2.13%～3.84%;对151例临床疑似样本的检出率为54.9%(83/151)。随机选择15份阳性样本PCR产物克隆测序,结果证明扩增片断为ALV的特异序列,与ALVgag基因核苷酸同源性为97.8～98.9%;对重庆14个县的14个健康商品蛋鸡群的134份粪便棉拭子进行检测,ALV的检出率为38.1%,鸡群的阳性率为14/14。同时用实时RT-PCR方法和ELISA方法对从种鸡场随机采集的30份100日龄蛋鸡的血清进行检测,实时RT-PCR检测率为5/30,ELISA方法检测率为4/30。本方法的建立为禽白血病的快速诊断、大规模检疫、监测、流行病学调查以及药物筛选等提供了新的检测方法。  相似文献   

11.
OBJECTIVE: To compare sensitivity and specificity of various polymerase chain reaction (PCR) assays for detection of Leptospira borgpetersenii serovar hardjo in bovine urine and to compare results of the optimal PCR assay with results of immunofluorescence, nucleic acid hybridization, and bacteriologic culture. ANIMALS: 6 heifers. PROCEDURE: Heifers were exposed to serovar hardjo type hardjo-bovis by conjunctival instillation of 10(6) leptospires on 3 successive days. Urine samples were collected before and after infection. Sensitivity and specificity of 5 PCR assays were compared, to determine the optimal assay for use with bovine urine samples. The optimal PCR assay was then compared with results of bacteriologic culture, nucleic acid hybridization, and immunofluorescence. RESULTS: A PCR assay with the best combination of specificity (100%) and sensitivity (91%) was selected for comparison with the other diagnostic tests. Sensitivity for nucleic acid hybridization was 55%, whereas sensitivity for bacteriologic culture and immunofluorescence was 89 to 93%. CONCLUSIONS AND CLINICAL RELEVANCE: Bacteriologic culture, PCR, and immunofluorescence were sensitive for detection of L borgpetersenii serovar hardjo type hardjo-bovis in urine specimens of cattle, but a single technique was not the most sensitive for each animal tested. Therefore, the use of 2 techniques in combination is warranted for maximal sensitivity for diagnosis.  相似文献   

12.
A polymerase chain reaction (PCR) assay was developed for the generic detection of Salmonella sp. and the identification of S. Enteritidis (SE), S. Gallinarum (SG), S. Pullorum (SP) and S. Typhimurium (ST) in material collected in the field from poultry. The specificity and sensitivity of the assay combined with Rappaport-Vassiliadis selective enrichment broth (PCR-RV) were determined, and field samples were analyzed to verify the validity of the method application. Specificity of the assay was tested using 29 SE, 11 SG, 10 ST and 10 SP strains, along with 75 strains of 28 other Salmonella serovars and 21 strains of other bacterial genera. The assay was 100% specific for Salmonella detection and ST identification. The primer pair for SE, SG and SP also detected S. Berta. PCR detection limits for Salmonella at the genus level were 2 ST, 8 SE, 1.1x10(3) SG and 1.8x10(5) SP cells. At the serovar level, detection limits were 7 ST, 1.2x10(3) SE, 4.4x10(7) SG and 1.8x10(6) SP cells. At the genus level, PCR-RV detected approximately 128% more positive field samples than the standard microbiological techniques and results were ready in 48h instead of 7 days. PCR-RV method is diagnostic of Salmonella at the genus level and ST at the serovar level, although other tests are needed to identify SE, SG and SP to serovar level.  相似文献   

13.
This study was performed to compare acid-phosphatase staining with polymerase chain reaction (PCR) analysis for the diagnosis of Dirofilaria repens infection. The infection of D. repens was confirmed in Korean reared German shepherd dogs. Knott's tests were carried out for the detection of microfilaria in 543 Korean reared German shepherd dogs (255 females and 288 males). Eighty four of the 543 dogs (15.5%) showed microfilaria-positive reactions with the modified Knott's test, and the test-positive microfilariae were then examined by both acid phosphatase staining and PCR analysis. Six (7.1%) and 17 (20.2%) of the 84 microfilaria-positive samples, by the Knott's tests were positive to D. repens by acid-phosphatase staining and in D. repens-specific PCR analysis, respectively. All samples found to be positive by the acid-phosphatase staining were also found to be positive by PCR analysis. Therefore, we conclude that PCR analysis (20.2%) is more valuable for the diagnosis of D. repens infection than acid-phosphatase staining (7.1%) (p<0.001).  相似文献   

14.
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15.
Reasons for performing study: Equine rhinitis viruses (ERV) cause respiratory disease and loss of performance in horses. It has been suggested that the economic significance of these viruses may have been underestimated due to insensitive methods of detection. Objectives: To develop a sensitive, rapid, real‐time RT‐PCR (rRT‐PCR) assay suitable for the routine diagnosis and epidemiological surveillance of the A and B variants of ERV. Methods: TaqMan primer probe sets for ERAV and ERBV were designed from conserved regions of the 5′ UTR of the ERV genome. Over 400 samples from both clinically affected and asymptomatic horses were employed for validation of the assays. ERAV samples positive by rRT‐PCR were verified by virus isolation and ERBV positive samples were verified by rRT‐PCR using a different set of primers. Results: The detection limit of the rRT‐PCR for both viruses was 10–100 genome copies. Of 250 archival nasal swabs submitted for diagnostic testing over a 7 year period, 29 were ERAV positive and 3 were ERBV positive with an average incidence rate per year of 10 and 1.5%, respectively. There was evidence of co‐circulation of ERAV and ERBV with equine influenza virus (EIV). Of 100 post race urine samples tested, 29 were ERAV positive by rRT‐PCR. Partial sequencing of 2 ERBV positive samples demonstrated that one was 100% identical to ERBV1 from a 270 bp sequence and the other was more closely related to ERBV2 than ERBV1 (95% compared to 90% nucleotide identity in 178 bp). Conclusions: The rRT‐PCR assays described here are specific and more sensitive than virus isolation. They have good reproducibility and are suitable for the routine diagnosis of ERAV and ERBV. Potential relevance: These assays should be useful for investigating the temporal association between clinical signs and rhinitis virus shedding.  相似文献   

16.
根据禽网状内皮组织增生症病毒(REV)长末端重复序列LTR基因保守区域设计并合成一对引物,建立基于SYBR GreenI模式的实时荧光PCR方法(Real-timePCR)。以常规PCR产物为标准品建立标准曲线,并对方法的特异性、敏感性、可重复性以及再现性、检出率进行评价。结果表明,该方法只从REV阳性样本检出扩增信号,不与其它病原出现交叉反应;熔解温度为86.8±0.5℃,无引物二聚体;扩增产物片段大小为270bp,与预期大小相符,测序结果证实为REV靶序列;在1.01×102～1.01×109拷贝之间呈现良好的线性关系,相关系数为0.999,扩增效率为99.7%,最低可检测101拷贝/反应阳性标准品,比常规PCR敏感103倍;组内变异系数、组间变异系数分别为4.80%～5.72%、0.75%～3.87%;对16例临床疑似病例进行检测,阳性率为81.25%(13/16),随机抽取7份阳性扩增产物进行测序,证实为REV序列。本研究建立的SYBR GreenI实时荧光PCR特异性强、通量高、灵敏度高、检测速度快,为REV的快速检测、分子流行病学调查以及监测REV污染疫苗情况提供新方法。  相似文献   

17.
Genetic aberrations linked to tumorigenesis have been identified in both canine and human hematopoietic malignancies. While the response of human patients to cancer treatments is often evaluated using cytogenetic techniques, this approach has not been used for dogs with comparable neoplasias. The aim of this study was to demonstrate the applicability of cytogenetic techniques to evaluate the cytogenetic response of canine leukemia to chemotherapy. Cytology and flow cytometric techniques were used to diagnose chronic myelomonocytic leukemia in a dog. High‐resolution oligonucleotide array comparative genomic hybridization (oaCGH) and multicolor fluorescence in situ hybridization (FISH) were performed to identify and characterize DNA copy number aberrations (CNAs) and targeted structural chromosome aberrations in peripheral blood WBC at the time of diagnosis and following one week of chemotherapy. At the time of diagnosis, oaCGH indicated the presence of 22 distinct CNAs, of which trisomy of dog chromosome 7 (CFA 7) was the most evident. FISH analysis revealed that this CNA was present in 42% of leukemic cells; in addition, a breakpoint cluster region‐Abelson murine leukemia viral oncogene homolog (BCR‐ABL) translocation was evident in 17.3% of cells. After one week of treatment, the percentage of cells affected by trisomy of CFA7 and BCR‐ABL translocation was reduced to 2% and 3.3%, respectively. Chromosome aberrations in canine leukemic cells may be monitored by molecular cytogenetic techniques to demonstrate cytogenetic remission following treatment. Further understanding of the genetic aberrations involved in canine leukemia may be crucial to improve treatment protocols.  相似文献   

18.
A Tritrichomonas foetus-specific 5' Taq nuclease assay using a 3' minor groove binder-DNA probe (TaqMan MGB) targeting conserved regions of the internal transcribed spacer-1 (ITS-1) was developed and compared to established diagnostic procedures. Specificity of the assay was evaluated using bovine venereal microflora and a range of related trichomonad species. Assay sensitivity was evaluated with log(10) dilutions of known numbers of cells, and compared to that for microscopy following culture (InPouch TF test kit) and the conventional TFR3-TFR4 PCR assay. The 5' Taq nuclease assay detected a single cell per assay from smegma or mucus which was 2500-fold or 250-fold more sensitive than microscopy following selective culture from smegma or mucus respectively, and 500-fold more sensitive than culture followed by conventional PCR assay. The sensitivity of the conventional PCR assay was comparable to the 5' Taq nuclease assay when testing purified DNA extracted from clinical specimens, whereas the 5' Taq nuclease assay sensitivity improved using crude cell lysates, which were not suitable as template for the conventional PCR assay. Urine was evaluated as a diagnostic specimen providing improved and equivalent levels of T. foetus detection in spiked urine by both microscopy following culture and direct 5' Taq nuclease detection, respectively, compared with smegma and mucus, however inconclusive results were obtained with urine samples from the field study. Diagnostic specimens (n=159) were collected from herds with culture positive animals and of the 14 animals positive by 5' Taq nuclease assay, 3 were confirmed by selective culture/microscopy detection (Fisher's exact test P<0.001). The 5' Taq nuclease assay described here demonstrated superior sensitivity to traditional culture/microscopy and offers advantages over the application of conventional PCR for the detection of T. foetus in clinical samples.  相似文献   

19.
Culturing of Mycobacterium avium subspecies paratuberculosis (Map) remains difficult and is time consuming. An alternative for the rapid detection of Map in samples is PCR. We have developed a sensitive DNA-extraction method based on sequence capture for the rapid detection of M. avium subspecies paratuberculosis by PCR in fecal and tissue samples. The method detected 10(2)Map/g feces using spiked samples, and reached a diagnostic sensitivity of 33,7% compared to 22% for culture. Analysis of tissue samples gave 65 polymerase chain reaction (PCR)-positive (42.2%) and 49 culture-positive samples (31.8%). Therefore, the detection limit of the DNA-extraction is the same as previously reported for culture, the PCR assay could detect more positive samples than the culture method.  相似文献   

20.
Lin Z  Zhang Y  Zhang H  Zhou Y  Cao J  Zhou J 《Veterinary parasitology》2012,185(2-4):296-300
Loop-mediated isothermal amplification (LAMP) is a simple method that can amplify DNA with high specificity, sensitivity, and rapidity. In this study, we compared the performance of LAMP and real-time PCR assays for diagnosis of toxoplasmosis. We designed a real-time PCR assay targeting a 529 bp element repeated 200-300 times in the Toxoplasma gondii genome. The detection limits of the LAMP and real-time PCR assays were 10 fg/μL and 1 fg/μL of T. gondii DNA, respectively. Conventional PCR, LAMP, and real-time PCR methods were applied to detect T. gondii DNA in blood samples from 284 pigs and 292 sheep. Positive results were obtained with 0.4%, 3.2%, and 4.2% of the pig samples and 3.8%, 17.1%, and 17.8% of the sheep samples with conventional PCR, LAMP, and real-time PCR analyses, respectively. The real-time PCR assay provided the most sensitive diagnosis of toxoplasmosis, but the LAMP assay has potential as an alternative tool for detection of T. gondii in the field.  相似文献   

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