Effects of Ureaplasma diversum on bovine oviductal explants: quantitative measurement using a calmodulin assay.

Calmodulin (CAM) acts as an intracellular regulator of calcium, an important mediator of many cell processes. We used the CAM assay and electron microscopy to investigate the effects of Ureaplasma diversum on bovine oviductal explants obtained aseptically from slaughtered cows. A stock suspension of U. diversum (treated specimens) and sterile broth (controls) was added to replicates of cultured explants and incubated at 38 degrees C in an atmosphere of 5.5% CO2 for 48 hours. Explants were examined for ciliary activity, extracellular CAM loss, and for histological and ultrastructural changes. Explants and their culture media were examined for changes in CAM concentration. All experiments were replicated three times. In addition, U. diversum, medium and broth were assayed for CAM content. The concentrations of CAM in explants and media changed significantly (p < 0.05) in samples which were inoculated with U. diversum when compared to controls. The controls and infected specimens did not differ histologically or ultrastructurally, but U. diversum was seen to be closely associated with infected explant tissue. In view of this close affinity it is assumed the loss of CAM from the oviductal cells was causally related, but this was not proven. The failure to show cell membrane injury on light and electron microscopic examination was probably related to the short duration of the experiment and may only point out the sensitivity of the CAM assay in detecting early cell membrane injury. Compromise in characteristics of the medium to support both, the viability of oviductal cells and U. diversum limited the experimental time to 48 hours.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro exposure of bovine morulae to Ureaplasma diversum.

Ureaplasma diversum has been associated with infertility in the cow experimentally and in naturally occurring cases. However, the pathogenic mechanism is undetermined. The purpose of this study was to determine whether ureaplasmas are pathogenic for bovine morulae in vitro. Twenty-one morulae were recovered from three superovulated, mature, Holstein cows six or seven days postestrus. The embryos were divided into three groups (A,B,C) and incubated for 16 hours at 37 degrees C in humidified air with 10% CO2. Group A was incubated in embryo culture medium alone, Group B was incubated in culture medium with sterile ureaplasma broth added and Group C was incubated in culture medium containing 1.7 X 10(6) colony forming units Ureaplasma diversum strain 2312. After incubation, the morulae were examined using an electron microscope. Structures morphologically identical to U. diversum were present on the outer surface of the zonae pellucidae of all the morulae exposed to the organism and none were present on the unexposed control embryos. No other morphological differences were observed in either the ureaplasma-exposed embryos or the two groups of control embryos. Ureaplasma diversum was isolated from three of the five embryos incubated in culture medium with sterile ureaplasma broth added. These three embryos were recovered from one donor cow which cultured positive for U. diversum from the vulva and flush fluid. This finding suggests that the contaminating organisms entered the embryo culture wells either in the embryo collection medium or attached to the embryos.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of murine macrophages and lymphocytes by Ureaplasma diversum.

Ureaplasma diversum is a pathogen in the bovine reproductive tract. The objective of the research was to study interactions with macrophages and lymphocytes which might elucidate aspects of pathogenetic mechanisms of this organism. We studied the activation of murine macrophages of C3H/HeN (LPS-responder) and C3H/HeJ (LPS-low-responder) genotype for TNF-alpha, IL-6, IL-1 and nitric oxide production and blastogenic response of C3H/HeJ splenocytes after Ureaplasma diversum stimulation. Live and heat-killed U. diversum induced TNF-alpha, IL-6 and IL-1 in peritoneal macrophage cultures of both C3H/HeN and C3H/HeJ mice in a dose dependent manner. Interferon-gamma modulated the cytokine production, by increasing the production of TNF-alpha, IL-6 and nitric oxide, but IL-1 secretion was only enhanced in C3H/HeJ macrophages stimulated by live ureaplasmas. Supernatant of U. diversum sonicate was mitogenic for murine spleen lymphocytes. The blastogenic response was dose dependent, and stimulation with both U. diversum and Concanavalin A seemed to have an additive effect. These results suggest that U. diversum, similar to other mycoplasmas, activates murine macrophages and lymphoid cells. The studies should be repeated with bovine cells in order to elucidate pathogenetic aspects of inflammation in cattle caused by U. diversum.     

Detection of Brucella melitensis in semen using the polymerase chain reaction assay

An evaluation of the polymerase chain reaction (PCR) for detection of Brucella melitensis DNA in bovine and ovine semen was performed. Since semen contains different components that inhibit PCR amplification, a protocol was used to purify Brucella-DNA from bovine and ovine semen samples prior to conducting amplification of the targeted DNA. When separated fractions of naturally Brucella contaminated semen were analyzed by the PCR, most of B. melitensis DNA were present in the seminal fluid and non-sperm fractions.The PCR examination results for detection of B. melitensis DNA in different semen fractions were compared with the results for traditional cultural methods of Brucella from semen. The PCR was more sensitive than the traditional cultural methods since it detected Brucella-DNA in 12 (10%) out of 120 semen samples while direct culture detected only 7 (5.8%) in the same semen samples. The limit of detection by PCR was 100 CFU/ml of semen. In addition, the results of PCR were available in one day, whereas isolation and identification of Brucella organisms required days or even weeks. The PCR may be used as a supplementary test for detection of B. melitensis in semen.     

The effect of intrauterine inoculation with Ureaplasma diversum on bovine fertility.

To determine the influence of Ureaplasma diversum on bovine fertility 11 uninfected virgin heifers with normal ovarian cyclic activity were randomly allocated to test or control groups. At a synchronized estrus, five test heifers were given an intrauterine broth inoculum containing 1.09 x 10(8) to 1.4 x 10(9) colony forming units of U. diversum and six control animals were infused with sterile ureaplasma broth medium. All animals were artificially inseminated within one hour of infusion. Pregnancy was diagnosed in one of five test heifers and all of six controls by serum progesterone concentrations measured to 25 days postinsemination. The difference in pregnancy rates between the two groups was statistically significant (p = 0.0152). It was concluded that under the conditions of this experiment U. diversum is capable of causing infertility in cattle.     

Development of a polymerase chain reaction assay for the detection of antibiotic resistance genes in community DNA.

Many methods are used to detect antibiotic resistance genes in samples. The objective of the study reported here was to compare polymerase chain reaction (PCR) analysis of community DNA with fecal culturing for detecting antibiotic resistance genes in cattle samples. In the laboratory-based portion of this study, known concentrations of an Escherichia coli strain with 3 antibiotic resistance genes (cmy-2, flo, and cat) were added to feces from dairy cattle. These genes were used to assess the effect of various primer pairs, chromosomally versus plasmid-encoded genes, and gene copy number on the sensitivity of PCR amplification. Gene-specific PCR amplification was performed on the community DNA extracted from the feces. Feces were cultured for the inoculated strain. In the field-based portion of the study, 80 cattle fecal samples of unknown gene status were compared by use of similar methods. Culture and PCR amplification from community DNA extractions produced variable results, and this variability was most noticeable at dilutions that approached the detection limit of the assay. Typically, PCR amplification had a higher sensitivity than did culture for detecting the gene of interest. However, the sensitivity of culture was improved by plating on selective media containing antibiotics. The community DNA approach enables assessment of bacterial communities in complex samples such as feces, a task that can be prohibitive by budget or time constraints associated with culture methods. Through a strategic combination of culture and community DNA approaches, the relationship between specific selection pressures and the persistence and dissemination of specific resistance genes can be elucidated.     

Comparison of polymerase chain reaction assays with bacteriologic culture, immunofluorescence, and nucleic acid hybridization for detection of Leptospira borgpetersenii serovar hardjo in urine of cattle

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.     

牛分枝杆菌特异性PCR检测方法的建立及初步应用

根据已发表的牛分枝杆菌的pncA的基因序列，设计和合成了一对可扩增294bp目的片段的引物，建立了特异性检测牛分枝杆菌的PCR方法。对牛分枝杆菌国际参考株和国内分离株成功扩增出294bp的特异性基因片段；对人结核分枝杆菌、副结核分枝杆菌、鸟胞内分枝杆菌和草分枝杆菌DNA的PCR扩增结果均为阴性。本PCR方法检测的敏感度可达到50pg。对10份牛分枝杆菌培养阳性和10份阴性样品的DNA分别进行了PCR检测，结果10份阳性样品中有9份样品为PCR扩增阳性，阳性符合率为90％（9／10）；而10份阴性样品则PCR扩增全部为阴性，阴性符合率为100％（10／10）。本方法可做为牛分枝杆菌的快速检测和流行病学调查的工具。     

Rapid PCR detection of Salmonella in horse faecal samples

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.     

Serogrouping of United States and some African serotypes of bluetongue virus using RT-PCR

The diagnostic potential of RT-PCR for detection of bluetongue virus (BTV) ribonucleic acid (RNA) sequence in cell culture and tissue samples from infected ruminants from United States, Sudan, South Africa and Senegal, was evaluated. The non structural protein 1 (NS1) gene of North American BTV serotype 11 was targeted for PCR amplification. The United States BTV serotypes 2, 10, 11, 13 and 17 and the Sudanese BTV serotypes 1, 2, 4 and 16 and BTV serotype 4 from South Africa and BTV serotype 2 from Senegal were studied. RNAs from all BTV field isolates used in this study, propagated in cell cultures, were detected by the described RT-PCR-based assay. The first specific 790bp BTV PCR products were amplified using a pair of outer primers (BTV1 and BTV2). Specificity of the PCR products was confirmed by a nested amplification of a 520bp PCR product using a pair of internal (nested) primers (BTV3 and BTV4). The BTV PCR products were visualized on ethidium bromide-stained agarose gels. Amplification products were not detected when the RT-PCR-based assay was applied to RNAs from closely related orbiviruses including, epizootic hemorrhagic disease virus (EHDV) prototypes serotypes 1, 2, 4; RNA from Sudanese isolate of palyam orbiviruses serogroup and total nucleic acid extracts from uninfected Vero cells. Application of the nested BTV RT-PCR to clinical samples resulted in amplification of BTV RNA from blood and serum samples from goats experimentally infected with BTV4 and from naturally infected sheep, goats, cattle and deer. The results of this study indicated that this RT-PCR assay could be applied for rapid detection of BTV, in cell culture and clinical samples from susceptible ruminants during an outbreak of the disease, in the United States and African.     

A polymerase chain reaction (PCR) assay for the detection of bovine herpesvirus 1 (BHV1) in selectively digested whole bovine semen

A PCR assay for the detection of bovine herpesvirus type 1 (BHV1) DNA in selectively digested whole bovine semen was developed and evaluated. A brief treatment with proteinase-K was used to lyse free virus, virus present in non-sperm cells and virus adhering to the spermatozoa. Genomic bovine DNA was not released by this treatment. Primers and probes were based on the nucleotide sequence of the gD gene. BHV1 virus-spiked split samples were used as positive controls and the PCR products were detected by eye in ethidium bromide-stained agarose gels. Sequentially collected non-extended semen samples from experimentally infected bulls were used to compare this assay with virus isolation. Of a total of 162 ejaculates, 51 were found positive by virus isolation, whereas PCR detected BHV1 DNA in 73. PCR detected BHV1 DNA for a longer period after infection and reaction. Apart from its superior sensitivity, this PCR assay also has the advantage of being a relatively simple procedure, providing results within 24 h.Abbreviations AI artificial insemination - BHV1 bovine herpesvirus type 1 - PCR polymerase chain reaction - TCID₅₀ tissue culture infective dose, 50%     

The effects of two Ureaplasma diversum strains on early pregnancy in heifers.

Two field isolates of Ureaplasma diversum spp. were used to infect heifers at the time of insemination in a preliminary study to observe the effect of infection on early pregnancy. M84-14c-1 was a field isolate from a bull's prepuce typed by immunofluorescence to be similar to U. diversum strain T-44 (Group C). M84-477c-4 was a field isolate from bovine semen typed by immunofluorescence to be similar to U. diversum strain T-288 (Group A). All three heifers infected with M84-477c-4 had a mild granular vulvitis at some time during the trial. None was pregnant when slaughtered 27 days after infection. The result of infection with M84-14c-1, a preputial isolate, was not consistent. One heifer had no infection and a normal pregnancy, one heifer was infected with an abnormal pregnancy, and one heifer was open but ureaplasmas were not detected until day 17 of the trial.     

A multiplex PCR for the detection of Brucella spp. and Leptospira spp. DNA from aborted bovine fetuses

Bovine brucellosis and leptospirosis are important causes of bovine abortion around the world. Both diseases can be serologically diagnosed, but many factors may cause false positive and negative results. Direct methods based on bacteriological isolation are usually employed, but they are difficult, time consuming and dangerous. Monoplex polymerase chain reaction (PCR) have been successfully described for the detection of Brucella spp. and Leptospira spp. Aiming at improvement in the direct diagnosis, a multiplex PCR (mPCR) for the detection of these agents in aborted bovine fetuses is described. The detection threshold of the mPCR was evaluated in experimentally contaminated bovine clinical samples using a conventional proteinase K/SDS or a boiling-based extraction protocols. The mPCR was applied to two groups of clinical samples: 63 episodes of bovine abortion and eight hamsters experimentally infected with Leptospira interrogans serovar pomona. Adopting microbiological isolation as reference, the test showed a sensitivity of 100% in both groups of clinical samples. Seven samples collected from bovine fetuses were Brucella spp. culture negative but showed positive results in mPCR. Regarding Leptospira spp. detection, similar results were observed in three bovine clinical samples. All hamsters infected with Leptospira were positive in both microbiological culture and mPCR. The boiling extraction protocol showed better results in some clinical samples, probably by the removal of PCR inhibitors by heat treatment. The high sensitivity, simplicity and the possibility of detection of both bacteria in a single tube reaction support the use of the mPCR described in the routine diagnosis.     

Comparison of dot blot hybridization, polymerase chain reaction, and virus isolation for detection of bovine herpesvirus-1 (BHV-1) in artificially infected bovine semen.

Bovine semen samples spiked with bovine herpesvirus 1 (BHV-1) were used to compare dot blot hybridization, polymerase chain reaction (PCR), and virus isolation for detection of BHV-1 in bovine semen. The PCR amplification used primers targeting the BHV-1 thymidine kinase gene and a nucleic acid releasing cocktail (GeneReleaser); the PCR product was used as the DNA probe in dot blot hybridization; virus isolation was done in primary bovine fetal testis (BFT) cell cultures. Semen diluted 1:20 in tissue culture medium had the least cytotoxicity and inhibition of viral cytopathic effects in BFT cells, allowing detection of 1 TCID50/100 microL of BHV-1 suspension by virus isolation. The presence of foreign DNA such as bovine sperm DNA or salmon sperm DNA increased the sensitivity of dot blot hybridization in detecting BHV-1, allowing detection of 20,000 TCID50/100 microL of neat semen. The inhibition of PCR amplification of BHV-1 DNA in bovine semen was eliminated by diluting the samples 1:20 in tissue culture medium. The best PCR amplification was obtained when semen was diluted 1:20 and when a reaction buffer of pH 9.0, with 1.0 mM MgCl2 was used. Under these conditions, the PCR followed by ethidium bromide staining of agarose gels could detect 1 TCID20/100 microL of sample, whereas PCR followed by Southern blot hybridization could detect 0.01 TCID50/100 microL of sample.(ABSTRACT TRUNCATED AT 250 WORDS)     

Establishment of conditions for the detection of bovine herpesvirus-1 by polymerase chain reaction using primers in the thymidine kinase region.

Polymerase chain reaction (PCR) for detection of bovine herpesvirus-1 (BHV-1) was developed and optimized using 22 bp sense and 20 bp antisense primers in the thymidine kinase (TK) coding region. The amplification product is 183 bp long. The PCR optimization was done using BHV-1 tissue culture supernate (BHV-1TCS), concentrated BHV-1 tissue culture supernate (cBHV-1TCS) and sucrose gradient purified BHV-1 (pBHV-1). The sensitivity of four methods of sample preparation which are standard DNA extraction, modified proteinase K (PK) digestion, GeneReleaserTM + 34 cycles or + 44 cycles, and boiling were compared with virus isolation (VI) using BHV-1TCS. The incorporation of 10% glycerol in the reaction mixture, the incubation in PK for 18 hours and predenaturation of samples and cooling in ice prior to PCR were essential for the amplification of BHV-1 DNA for samples prepared by standard DNA extraction and modified PK digestion. The preparation of samples by Gene-ReleaserTM, a proprietary nucleic acid releasing cocktail, showed 10 to 1,000-fold increase in sensitivity compared to standard DNA extraction and modified PK digestion. No amplification was observed in samples prepared by boiling. The sample preparation of BHV-1 LA strain by GeneReleaserTM showed sensitivity equivalent to virus isolation. The BHV-1 TK PCR using GeneReleaserTM has a detection limit of 1 picogram and 10 fentograms of purified BHV-1 DNA using ethidium bromide stained gel and Southern blot hybridization, respectively. It could detect viral DNA in 1,000 infected cells in a total suspension of 10,000 cells using either ethidium bromide stained gel or Southern blot hybridization.     

实验猴粪便样品中沙门氏菌LAMP检测方法的建立及应用简

A loop-mediated isothermal amplification (LAMP) assay was developed for detection of Salmonlla in fecal samples of experimental monkeys. According to the specific sequences (fimY) of Salmonlla in GenBank, one set of primers was selected and the reaction condition was optimized. The results showed that the detection limit of LAMP method was 1.35×10¹ and 1.35×10³ CFU/mL in Salmonella pure culture and clinical samples,respectively,which was the same as routine PCR. The amplification could complete in one hour, and the result could be distinguished by naked eyes. There was non-specific amplification of other pathogens. These results suggested that this LAMP assay was a simple and specific method for rapid detection of Salmonlla in fecal samples.     

An improved molecular assay for Tritrichomonas foetus

Tritrichomonas foetus (T. foetus) is the causative agent of bovine trichomonosis, a sexually transmitted disease leading to abortion (from 1 to 8 months gestation), infertility, and occasional pyometra. The annual losses to the U.S. beef industry are estimated to be in the hundreds of millions of dollars. Currently, the "gold standard" diagnostic test for trichomonosis in most countries is the cultivation of live organisms from reproductive secretions. The cultured organisms can then be followed by PCR assays with primers that amplify T. foetus to the exclusion of all other trichomonad species. Thus, negative results present as null data, indistinguishable from failed PCR amplification during T. foetus specific amplification. Our newly developed assay improves previously developed PCR based techniques by using diagnostic size variants from within the internal transcribed spacer 1 (ITS1) region that is between the 18S rRNA and 5.8S rRNA subunits. This new PCR assay amplifies trichomonad DNA from a variety of genera and positively identifies the causative agent in the bovine trichomonad infection. This approach eliminates false negatives found in some current assays as well as identifying the causative agent of trichomonad infection. Additionally, our assay incorporates a fluorescently labeled primer enabling high sensitivity and rapid assessment of the specific trichomonad species. Moreover, electrophoretic separation of amplified samples can be outsourced, thus eliminating the need for diagnostic laboratories to purchase expensive analysis equipment.     

Comparison of faecal culture and IS900 PCR assay for the detection of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> in bovine faecal samples

Comparative efficacy of faecal culture and IS900 Polymerase chain reaction (PCR) assay of faecal samples was investigated in 40 clinically suspected cases of Johne’s disease in dairy cattle. The sensitivity of faecal culture and PCR assay in this study was 52.5% (21/40) and 90% (36/40) respectively. All isolates appeared only on the mycobactin J supplemented Herrold’s egg yolk medium (HEYM) at 8–16 weeks post-inoculation, were acid-fast and were positive for IS900 PCR yielding a single amplicon of 217 bp. A total of 28 faecal samples out of 40 were positive by IS900 primary PCR assay for Mycobacterium avium subsp. paratuberculosis (Map) yielding an expected product of size 217 bp. Twelve faecal samples, which gave negative results in the primary PCR, were subjected to secondary PCR assay. Of the 12 samples, 8 gave positive results in the IS900 nested PCR (nPCR), which yielded a PCR product of 167 bp, proving better sensitivity of nPCR assay than single amplification PCR. PCR could detect additionally 15 samples as positive which were negative by faecal culture. The chi-square analysis showed a highly significant difference between the tests (P< 0.01). This study suggests that IS900-PCR-based detection of Map could be used as a potential diagnostic tool for rapid and effective Johne’s disease surveillance.     

布鲁菌Cycling探针荧光定量PCR检测方法的建立

根据布鲁菌BCSP31基因序列设计布鲁菌通用检测引物和探针,建立了布鲁菌Cycling探针荧光定量PCR检测方法。以构建的含BCSP31基因的质粒标准品10倍递进稀释为模板检测其敏感性,结果显示,本方法能检测约10个拷贝的阳性质粒,且标准曲线的线性关系良好。用本方法检测5株不同种的布鲁菌以及猪大肠杆菌K99、巴氏杆菌C48-1、猪链球菌ST171、绿脓杆菌等4株对照菌。结果显示,5株不同种的布鲁菌均出现典型的"S"型扩增曲线,4株对照菌40个循环内均无CT值出现。用本方法和B4/B5-PCR方法对来自布鲁菌病流行地区3个不同牛场的40份血样、奶样和血清样进行平行检测。结果显示,本方法和B4/B5-PCR方法的结果符合率为80.0%。B4/B5-PCR检测为阳性的27份样品经本方法检测均为阳性;B4/B5-PCR检测为阴性的13份样本,经本方法检测,其中8份呈阳性,5份为阴性。本方法的敏感性明显高于B4/B5-PCR方法。试验表明,所建立的Cycling探针荧光定量PCR方法具有敏感、特异、稳定等特点,可用于布鲁菌感染的快速检测。