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)     

Humoral and secretory antibodies to Ureaplasma diversum in heifers following subcutaneous vaccination and vaginal infection.

We measured antibody levels in serum and cervicovaginal mucus (CVM) of four heifers vaccinated with two inoculations of killed Ureaplasma diversum strain 2312 in incomplete Freund's adjuvant (IFA) two weeks apart, and six heifers given a placebo. Two weeks later, the vaccinates and four placebo heifers, were challenged by intravaginal inoculation with 6.4 x 10(8) colony-forming units of the homologous U. diversum strain. The remaining two placebo heifers served as unvaccinated, unchallenged controls. Antibody levels in serum and CVM of all heifers were determined by an enzyme-linked immunosorbent assay (ELISA). Vaccination stimulated specific IgG1 and IgG2 responses in serum and CVM but only a slight IgM and no IgA response. In both vaccinate and placebo heifers, subsequent intravaginal challenge resulted in a granular vulvitis (GV) with a predominant IgA response in the CVM. The GV gradually subsided during the 35 day observation period but ureaplasmas were consistently demonstrated by culture. We concluded that subcutaneous vaccination stimulated a specific, albeit nonprotective, IgG response in serum and CVM. In contrast, vaginal infection primarily induced a mucosal IgA response.     

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.     

Adherence of bovine viral diarrhea virus to bovine oocytes and embryos with a hardened zona pellucida cultured in vitro

The purpose of this study was to investigate the adherence of bovine viral diarrhea virus (BVDV) to bovine mature, or immature, cumulus-free oocytes and to in vitro fertilized embryos, maintained in vitro in a ligated bovine oviduct to allow for the hardening of the zona pellucida. Incubation of the oocytes and embryos in the oviduct for 5 h caused hardening of the zona pellucida as measured by resistance to pronase digestion (which increased from approximately 3 min to 7 h; P >0.001). However, there was no difference between the number of infected oocytes and embryos (n = 965 in 193 samples) following experimental exposure to BVDV regardless of whether or not they were previously incubated in the oviduct (P > 0.05). It was concluded that the modification of the proteolytic resistance properties of the zona pellucida during in vitro oviductal incubation did not influence the adherence of BVDV to zona pellucida of oocytes or in vitro fertilized embryos.     

Relationship between plasminogen activator production and bovine embryo development in vitro

The relationship of plasminogen activator (PA) production to cell stage, cell number and changes in overall diameter and zona pellucida thickness for bovine embryos developing in vitro was determined. Late morulae to blastocysts (n = 80) were collected nonsurgically from naturally mated, estrous-synchronized, superovulated crossbred beef cows. Embryos were cultured, one embryo per 25-microliters microdrop, for 6 d. At 24-h intervals, embryos were evaluated for stage of development and transferred to fresh microdrops; media were recovered for PA analysis. In addition, embryo diameter and zona pellucida thickness were measured with an ocular micrometer. Plasminogen activator production was determined using a caseinolytic assay with urokinase as the standard. Changes in diameter, zona pellucida thickness and PA production per 24-h interval for each embryo were plotted, and the graphs were cut out and weighed. Sixty-one embryos (76%) completed the hatching process. Total PA production was correlated positively (P less than .005) to embryonic size (r = .40), developmental stage (r = .35) and cell number (r = .35) and negatively, but weakly, correlated to zona pellucida thickness (r = -.13; P = .267). Hatched embryos produced more total PA than embryos that did not hatch (.140 +/- .011 vs .070 +/- .019 g; P less than .01). These results suggest that as embryonic size and cell number increase and development progresses, bovine embryos liberate more PA.     

Comparison of persistence of seven bovine viruses on bovine embryos following in vitro exposure

The ability of seven cytopathic strains of bovine viruses to adhere to the zona pellucida of six-to-eight day-old bovine embryos were compared. Embryos were exposed to virus by placing them either in virus suspensions or by culturing them on infected bovine turbinate cultures for 18-24 h. After exposure to bovine virus diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBV), bluetongue virus (BTV), pseudorabies virus (PRV), vesicular stomatitis virus (VSV), parainfluenza 3 virus (PI3), or bovine enterovirus virus (BEV), the embryos were tested for virus by culture in bovine turbinate cells and by morphological examination using electron microscopy (EM). A special technique to minimize loss of embryos processed for EM was developed. More embryos had viral particles on the surface of the zona pellucida after exposure to 18-24 hour infected cell cultures than did embryos exposed to viral culture suspensions. The most dramatic finding was that BTV adhered in large numbers to the surface of the zona pellucida of exposed embryos. IBRV, PRV, and VSV comprised an intermediate group, with virions occasionally detected on the surface of exposed embryos after 5 washes. Therefore, extensive washing is required. The PI3 and BEV were easily removed from embryo-exposed virus by washing. BVD was difficult to identify morphologically, making assessment by EM unreliable. There was no evidence that any one of the seven viruses penetrated the intact zona pellucida. Using a micromanipulator, 42 embryos were also directly inoculated through the zona pellucida with +/- 50 picoliters of virus inoculum or medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Embryo transfer: a discussion on its potential for infectious disease control based on a review of studies on infection of gametes and early embryos by various agents

Studies on laboratory animals have shown that viruses vary as to whether or not they are transmissible by the gametes or are capable of passing through the zona pellucida and infecting the embryo.

Methods of studying early embryos for the presence of infectious agents include electron microscopy, immunocytochemistry and cell cultivation.

Determination that early bovine embryos do not become infected by certain agents might allow for easing of restrictions in the current import and export regulations for cattle embryos.

Embryo transfer could be used as a means of controlling or eliminating disease in a herd or flock if the causal agent does not infect the early embryo via the gametes or by penetrating the zona pellucida.

     

Immune response of heifers to vaginal submucosal or subcutaneous vaccination and intravaginal challenge with Ureaplasma diversum.

Twenty beef heifers were randomly assigned to five equal groups and vaccinated: Group 1--in vaginal submucosa (VM) with Ureaplasma diversum ultrasonicated whole cells (WC) in complete Freund's adjuvant (CFA); Group 2--in VM with U. diversum cell membranes (CM) in CFA; Group 3--subcutaneously (SC) with CM in CFA; Group 4--in VM with CM alone; and Group 5--in VM with phosphate buffered saline (PBS) in CFA. A second vaccination with the same antigens in incomplete Freund's adjuvant was given after four weeks, and three weeks later, all heifers were challenged intravaginally with 3.6 x 10(7) colony-forming units (CFU) of U. diversum strain 2312. Immunoglobulins that reacted with U. diversum were measured in serum and cervicovaginal mucus (CVM) by an enzyme-linked-immunosorbent assay. In groups 1 and 2, vaccination by the VM route with WC or CM antigens, stimulated high levels of U. diversum-reactive IgG1 and IgG2 antibodies in serum as well as CVM, but a low IgA response only in CVM. In group 4, VM vaccination with CM (no adjuvant) elicited a minimal IgG1 and IgG2 response in serum and CVM. In group 3, SC vaccination with CM antigen stimulated high IgG1 and IgG2 reactivity in both serum and CVM, but no IgA reactivity. Very little IgM reactivity was detected in the four vaccinated groups. Intravaginal challenge resulted in characteristic granular vulvitis in all vaccinated and control heifers, with all animals remaining culture-positive for the 35 day observation period. The infection stimulated a marked increase in the specific IgA response in CVM of the three groups vaccinated with either, adjuvanted antigen.(ABSTRACT TRUNCATED AT 250 WORDS)     

腺病毒介导的高效生产水牛转基因胚胎条件的摸索

试验旨在探究腺病毒感染水牛颗粒细胞和胚胎的最佳条件,以提高转基因水牛胚胎的生产效率。以水牛颗粒细胞和体外发育的胚胎为研究对象,分别用0、1×10~0、1×10^-1、1×10^-2、1×10^-3、1×10^-4、1×10^-5 GFU/mL腺病毒感染水牛颗粒细胞,获得最佳感染浓度后,在最佳浓度条件下分别感染24、48、72、96 h,摸索最佳感染时间,用倒置荧光显微镜观察试验结果。利用腺病毒感染水牛颗粒细胞的最佳浓度和时间分别感染2细胞和4细胞期胚胎,对胚胎的最佳感染条件进行摸索,分析胚胎分裂率、囊胚率和转染囊胚率。结果显示,1×10^-2 GFU/mL浓度和48 h感染时间可获得水牛颗粒细胞的最佳腺病毒感染效率。腺病毒感染2细胞和4细胞期无透明带和非完整透明带胚胎后胚胎发绿光,而感染完整透明带胚胎后不发光,2细胞期胚胎感染后停止发育,4细胞期开始感染的非完整透明带胚胎可继续发育至囊胚。于4细胞期分别感染完整透明带组、非完整透明带组和无透明带组水牛胚胎,结果显示,非完整透明带转染组在囊胚率和囊胚转染效率上均优于其他组。综上,非完整透明带,1×10^-2 GFU/mL和48 h为感染浓度和时间,4细胞期为感染起始期的腺病毒介导的水牛转基因胚胎生产方法能够实现目标基因在水牛胚胎中的高效表达,从而达到提高转基因水牛胚胎生产效率的目的。     

Transfer of bovine demi-embryos with and without the zona pellucida

Bisected bovine embryos with or without the zona pellucida were transferred to recipients nonsurgically in five field trials. Embryos were collected from superovulated donors 6.5 to 7.5 d after estrus; only embryos of good and excellent quality were bisected. Demi-embryos were transferred either within a zona pellucida, without a zona pellucida, without a zona pellucida, or in the third and fourth trials, without a zona but embedded in 7% gelatin. Pregnancies were diagnosed at 44 to 68 d of gestation. In a preliminary trial, 9/29 zona pellucida-intact demi-embryos developed into fetuses compared with 1/10 zona pellucida-free demi-embryos (P greater than .1). The proportion of zona-free demi-embryos developing to fetuses was not significantly different from the zona-intact group in the second trial either, 24/49 and 5/19, respectively. In trial 3, the proportion of zona pellucida-free demi-embryos developing was 8/25; of zona-enclosed embryos, 29/88; and of zona-free demi-embryos embedded in gelatin, 8/22 (P greater than .1). Similarly, in the fourth trial the rate of development of zona-free demi-embryos to fetuses was 5/12, that of zona-enclosed embryos was 32/81, and that of zona-free demi-embryos embedded in gelatin was 3/12 (P greater than .1). In trial 5, survival of zona-enclosed demi-embryos to fetuses was 40/105, and of zona-free demi-embryos, 46/109 (P greater than .1). Except for trial 2, half of the demi-embryos were twinned, one to each uterine horn; twinning did not significantly affect the proportion developing to fetuses for any of the demi-embryo groups. It is concluded that placing post-compaction demi-embryos into the zona pellucida for transfer does not improve pregnancy rates significantly.     

Prospects for the use of embryos in the control of disease and the transport of genotypes

Transfer and low temperature storage of embryos are now proven techniques for a number of mammalian species. These techniques are useful in control of disease and in saving genotypes from infected animals. The place of embryos in the epidemiology of disease depends upon whether the causative organism can gain entry to the oocyte before or at fertilisation and on whether the young embryo can be invaded by organisms in the uterine environment. There is little evidence that important live-stock diseases are transmitted via gametes. The zona pellucida surrounding the embryo is an effective barrier against a number of important disease organisms; in some cases the embryo is susceptible once it has hatched from the zona pellucida. It is important therefore in considering the use of embryos in disease control, to ensure that virus is not attached to the surface of the zona pellucida from where it can infect the recipient and/or the embryo after hatching. Washing procedures have been devised together with the use of enzymes and antisera to remove virus from the surface of embryos. Some viruses enter pores and sperm tracks in the zona and removal of these may present a problem. African swine fever virus has been shown to resist removal by treatment with enzymes. There are no guidelines as to the likely interaction between a certain virus and embryos. Therefore each virus of interest must be tested to determine whether it can be transmitted via washed embryos. Nevertheless there are numerous instances of the use of embryo transfer to eradicate a specific disease or to save valuable genetic material from infected animals without transmitting disease.     

Transfer of Bisected Cattle Embryos within the Same Zona Pellucida*

Cattle embryos at the compact morula and blastocyst stages were bisected by two different methods. In some embryos the embryo bisection was performed outside of the zona pellucida and both halves were replaced into it. Other embryos were bisected within the zona pellucida and no pipetting of the halves was required. They were transferred to synchronized recipient cows and the pregnancy rates and twinning rates were evaluated.     

Uterine tubal cells remain uninfected after culture with in vitro-produced embryos exposed to bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV) has been isolated from washed and sonicated, in vitro-produced embryos, but the infectivity of BVDV associated with intact, developing, embryos has not been demonstrated. The objective of this study was to determine if a dose of BVDV infective for co-culture cells was associated with individual, developing embryos, following artificial exposure to the virus and washing. In 5 replicates, zona pellucida-intact, in vitro-produced embryos were assigned to a negative control embryo group, or were incubated in 10(5)-10(6) cell culture infective doses (50%, CCID50) per milliliter of a type I, noncytopathic (strain SD-1) BVDV for 2 h. Unexposed negative control embryos and exposed positive control embryos were washed, sonicated and assayed for BVDV using virus isolation with immunoperoxidase monolayer assay. Immediately or following cryopreservation, remaining virally-exposed, washed embryos were co-cultured individually with BVDV-negative cultures of bovine uterine tubal cells in a medium free of BVDV-neutralizing activity. After two days in culture, uterine tubal cells and embryos (including the zona pellucida) were separated and washed. The culture medium, uterine tubal cells and embryos were then assayed for BVDV. Bovine viral diarrhea virus was not isolated from any negative control embryo group, but was isolated from all positive control embryo groups. Although all uterine tubal cell populations were confirmed to be susceptible to BVDV, virus was never isolated from uterine tubal cells or embryos from post-exposure culture. In conclusion, although BVDV remains associated with washed in vitro-produced embryos, the virus associated with unsonicated embryos was not infective for uterine tubal cells in vitro.     

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.     

A method for the cytogenetic detection of early embryos in heifers

The objective of the study was to work out a simple and reliable method of the cytogenetic examination of bovine embryos. Whole embryos free of the zona pellucida, or parts of these embryos, were cultivated for five hours in a medium with 0.05 microgram colcemide per 1 millilitre. The embryos were hypotonized for 10 to 15 minutes in a mixture of medium and distilled water (1:2 ratio) at the temperature of 37 degrees C. Stepwise fixation was used: first in a mixture of acetic acid, methyl alcohol and water (1:4:5), followed by dropping a mixture of acetic acid and methyl alcohol (1:1) onto the embryo already on the microscope slide during microscopic control. The chromosomes were stained with a 3% solution of Giemsa dye for 10 minutes.     

Susceptibility of bovine naked 2- and 4-cell embryos and hatched blastocysts produced in vitro to infection with noncytopathogenic bovine viral diarrhea virus.

To investigate the susceptibility of early bovine embryos to noncytopathogenic bovine viral diarrhea virus (NCP BVDV), 2- and 4-cell embryos produced in vitro from which zona pellucida had been removed by pronase treatment, and hatched blastocysts were exposed to 10(6) TCID50/m/ of NCP BVDV No. 12 strain. The virus was detected in all embryo samples immediately prior to cultivation but not in the medium. After 24-hr culture, the virus was isolated from four media and two embryo samples in four experiments in the blastocyst group, and the viral antigen was demonstrated in the cytoplasm of the embryo cells by the immunofluorescent technique. By contrast, no virus was recovered from, or viral antigen detected in samples from the 2- and 4-cell embryo group in any of the experiments, even though they were exposed to the virus after removal of the zona pellucida. These findings suggest that 2- and 4-cell embryos are unlikely to be susceptible to NCP BVDV, but that blastocysts are capable of being infected with the virus. hatched blastocyst, noncytopathogenic bovine viral diarrhea virus.     

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)     

Detection of antibodies to mycoplasmas using an immunoenzyme method

Detection of the antibodies to the species Mycoplasma bovis in the serum and milk of dairy cows coming from a mastitis-infected herd is a good example of utilization of the ELISA immunoenzymologic method in the mycoplasmology. Examining the samples from 75 dairy cows and applying the indirect hemagglutination test, good correlation of the results of the two tests was determined. The antibodies to the species Ureaplasma diversum were demonstrated by the ELISA method both in the bovine serum and in the milk of dairy cows infected slightly with mastitis. We chosen that strain which detected the maximum titres in the selected samples of the sera out of four antigens prepared from various strains of U. diversum. Rabbit sera hyperimmune to 26 strains of the mycoplasmas of various species were used to identify two antigens (after removing the antibodies to the components of the media). Specific reaction was obtained with the antisera to M. hyorhinis and M. arginini.