Intrauterine Inoculation Research Articles

Early abortion in cattle induced by experimental intrauterine infection with pure cultures of Actinomyces pyogenes

Actinomyces pyogenes from a case of endometritis was used to study the effects of infection of the bovine embryo between days 27 and 41 of pregnancy. From 10(9) to 10(10) washed organisms were introduced into the uterine lumen of four pregnant cows. Two pregnant cows were inoculated with sterile saline and four pregnant cows were treated with cloprostenol. Embryonic death and abortion followed 29 to 144 hours after the inoculation of the live bacteria. The aborted embryos were macerated or clearly degenerating and yielded profuse pure cultures of A pyogenes. Abortion was accompanied by a sustained increase in uterine tone, opening of the cervix, presence of vaginal pus and a vulval discharge and the persistence of the corpus luteum for at least eight days after abortion. Intrauterine inoculation with saline did not affect pregnancy, but embryonic death, abortion and regression of the corpus luteum occurred 66 to 72 hours after the treatment with cloprostenol. The results suggest that A pyogenes is a primary pathogen and is capable of causing embryonic death and abortion.

Evaluation of pregnant rabbits as a laboratory model for bovid herpesvirus-4 infection

SUMMARY A field strain (87-8363) of bovid herpesvirus-4 (bhv-4) isolated from an aborted bovine fetus was used to inoculate pregnant rabbits. Eleven rabbits in midgestation were alloted to 4 groups consisting of 3 infected groups and 1 control group. Rabbits were inoculated with bhv-4 or mock-infected cell culture preparations via iv, intravaginal, and intrauterine routes. Mild vulvovaginitis and endometritis were observed after intravaginal and iv inoculation of bhv-4, whereas intrauterine inoculation of bhv-4 resulted in abortion of hemorrhagic fetuses and nonsuppurative endometritis. Virus was successfully isolated from organ explants of fetal tissues. Rabbits seroconverted 1 week after infection as detected by results of an indirect immunofluorescence assay.

Endometritis in the mare: A comparison between reproductive history and uterine biopsy as techniques for predicting susceptibility of mares to uterine infection

Thirty mares with no clinical signs of endometritis were categorized as being susceptible or resistant to uterine infection depending on whether or not they had a history of recurrent endometritis. The same mares were then independently classified as susceptible or resistant on the basis of their uterine biopsies; those with significant endometrial degeneration were considered to be susceptible to endometritis. The mares then received an intrauterine inoculation of pathogenic Streptococcus zooepidemicus . Those mares which eliminated bacteria by 10 d after inoculation were considered truly resistant to endometritis, whereas those still infected at 10 d were considered susceptible. The original classifications based on history or biopsy were compared to the inoculation results. A history of recurrent endometritis provided a more sensitive (0.90) and specific (0.95) indication of susceptibility to uterine infection than a uterine biopsy with significant endometrial degeneration (sensitivity 0.5, specificity 0.75).

Serological reactions in sheep and cattle experimentally infected with three Australian isolates of bluetongue virus

Serums from 103 sheep and 24 cattle experimentally infected with one of 3 serotypes of bluetongue virus isolated in Australia were tested for antibody to bluetongue virus in the serum neutralisation test and the agar gel diffusion precipitin test. Antibody to bluetongue virus was first detected by these tests 8 to 10 days after intravenous infection in 4 sheep that were bled daily for serum analysis. The agar gel diffusion test failed to detect antibody in 28% (29/103) of sheep which had seroconverted in the serum neutralisation test. A further 7% (7/103) of sheep serums were negative in both tests 14 to 22 d after infection. Both tests detected antibody to bluetongue virus in all cattle serums by 10 days after detection of viraemia. In comparison with the intravenous route of infection, extended prepatent periods for the commencement of viraemia resulting from intradermal, subcutaneous and intrauterine routes of infection in the cattle caused corresponding delays in the detection of antibody. For example, one cow that was infected by intrauterine inoculation did not become viraemic until 22 d after inoculation and antibody was not detected until 32 d after inoculation.

Effect of [formula omitted][formula omitted] and gram-negative anaerobic bacteria on the development of bovine pyometra

Fifteen lactating Holstein cows were used in a trial designed to evaluate the effectiveness of intrauterine inoculation (challenge) of Actinomyces pyogenes (A) alone or in combination with Fusobacterium necrophorum (F) and Bacteroides melaninogenicus (B) to induce pyometra. Cows were assigned to one of five groups: A (n = 3), AB (n = 3), AF (n = 3), ABF (n = 3) or C (control, broth medium alone; n = 3). All cows exhibited estrus 12 or 13 d prior to challenge (Day 0=first day of challenge). During the prechallenge period, the reproductive tract of each cow was palpated per rectum and uterine fluid aspirates for culture and uterine biopsies were also obtained. All cows received an intravenous injection of 5,000 IU of human chorionic gonadotropin (hCG; Day 5) and an intrauterine infusion of 40 ml of 0.7% iodine solution (Day 1). Cows were then inoculated on Days 0, 1 and 2 of the experiment. Sequential palpations of the reproductive tracts, samples of uterine fluid for culture and uterine biopsies were performed for a total of 30 d after the first inoculation. A cow was diagnosed as having pyometra when purulent uterine fluid and a corpus luteum were detected by palpation per rectum. The number of cows that developed pyometra in Group A was 2 of 3, in Group AB 3 of 3, in Group AF 3 of 3, in Group ABF 3 of 3 and in Group C 0 of 3. Cows with pyometra did not exhibit estrus. In 7 of 11 cows, pyometra persisted for more than 21 d. In cows that developed pyometra, the same species of bacteria infused into the uterus were usually recovered one or more times during the postchallenge period. When clinical pyometra was diagnosed, histologic endometritis was invariably present. Histologic endometritis and concurrent isolation of A . pyogenes alone or A . pyogenes with gram-negative anaerobic bacteria occurred in 91.7% of samples during the postchallenge period. Regardless of bacterial treatment, gram-negative anaerobic bacteria were frequently isolated with A . pyogenes during this period.

Evaluation of the uterine environment in experimental and spontaneous bovine metritis

This study describes a model for experimental induction of metritis in cattle. Cows were given two intrauterine inoculations of a mixture of Corynebacterium pyogenes and Bacteroides melaninogenicus at 12 and 36 h following calving. The endometrium was subjected to minor irritation just prior to inoculation with bacteria. All cows showed signs of severe metritis within 48 h following the last intrauterine inoculation. The study also evaluated intrauterine oxygen-reduction potential (E h) and pH following calving in control cows, cows with retained fetal membranes, and cows with induced metritis. The results revealed that cows with retained fetal membranes or induced metritis had lower E h values (P < 0.05) than control cows on Days 2, 3, or 4 postpartum. This suggests that antibiotics that are effective under anaerobic conditions should be considered for intrauterine therapy during this period.

An Outbreak of Septic Endometritis in the Arctic Blue Fox (Alopex Lagopus) caused by Clostridium Carnis

An outbreak of endometritis and septicaemia in farms that raise Arctic blue foxes is described. Eleven animals from eight different farms were examined, most of them pregnant or with newly delivered litters. Pathological examination indicated inflammation of the uterus, and focal necrotic changes in the liver. In some animals evidence of hemorrhaging was found in the anterior part of the small intestine. Bacteriological examination revealed abundant growth from different organs of the animals of a pure culture of a small, slender, Gram-variable, sporeforming rod, which was identified phenotypically as aerotolerant Clostridium carnis. This identification was confirmed by DNA hybridization. The organism caused death in mice 1–2 days after intraperitoneal inoculation, and killed an Arctic blue fox which was inoculated intracervically. Two foxes, pretreated with diethyl-stilboestrol, became seriously ill after intrauterine inoculation, but survived. The spontaneous outbreak of the disease was characterized by sudden onset of septicaemia with few or no clinical symptoms before the animals succumbed.

Embryonic loss in pony mares induced by intrauterine infusion of [formula omitted][formula omitted

Pony mares which were detected pregnant by transrectal ultrasonography received a single intrauterine infusion of either sterile saline (control, n = 12 mares) or 10 6 Candida parapsilosis (treated, n = 12 mares) between Days 11 to 14 postovulation. Subsequent embryonic loss was studied by daily ultrasonography of the mare's uterus, by serum progesterone levels, by endometrial swabs for cytologic and microbiologic examination and by endometrial biopsies that were taken after embryonic loss was detected. Significantly fewer (P<0.01) embryonic losses occurred in control than in treated mares ( 4 12 vs 12 12 ). The mean interval from intrauterine infusion until embryonic loss was 5.8 ± 2.8 d for control mares (n = 4) and 2.1 ± 0.2 d for treated mares (n = 12). Prior to embryonic loss, moderate to marked edema of the endometrial folds in 12 of 12 treated mares and free fluid in the uterine lumen of 5 of 12 treated mares were detected by ultrasonography. After embryonic loss, Candida parapsilosis was cultured from the uteri of 8 of 12 treated mares, and E . coli was cultured from the uteri of 2 of 4 control mares. Postloss endometrial smears had cytologic evidence of inflammation in 10 of 12 treated mares and 3 of 4 control mares. Intrauterine inoculation of C. parapsilosis consistently induced embryonic loss and may provide a basis to further study the relationship between endometritis and embryonic loss in mares.

Clearance of bacteria and non-antigenic markers following intra-uterine inoculation into maiden mares: Effect of steroid hormone environment

Uterine response to inoculation with Streptococcus zooepidemicus organisms (antigenic markers) and 15-μm microspheres and charcoal (non — antigenic markers) was determined in seasonally acyclic maiden mares treated with either progesterone (P) n = 4, estradiol (E) n = 4 or oil vehicle (C) n = 4. At 3, 7 and 15 d after inoculation with bacteria and the 2 non — antigenic markers, uteri were flushed and the clearance of these materials, as well as the number of white blood cells and immunoglobulin concentration, determined. P-treated mares had higher numbers of bacteria and IgA and a greater volume of purulent fluid in the uterus than E- or C-treated mares at 7 d after inoculation. Clearance of inoculated materials began within 2 h in E-treated mares, and the non-antigenic markers were completely cleared in E- and C-, but not in P-treated mares, in 3 d. This suggests that in the P-dominated uterus, reduced physical clearance may contribute to an increased susceptibility to uterine infection.

Experimental intrauterine inoculation of pregnant ewes with ureaplasmas.

The pregnancies of 13 ewes which were inoculated intrauterine with one of two strains of ovine ureaplasmas resulted in 9 normal and 3 abnormal births, and one ewe was found to be no longer pregnant on postmortem examination. Vaginal ureaplasma infection was detected in the majority of ewes only after lambing. Of the 12 ewes examined at postmortem, ureaplasmas were isolated from the uterus of 5 out of the 6 necropsied up to 21 days post-partum. The vulvar/preputial areas of the majority of lambs that survived were infected with ureaplasmas for the duration of the experiment, but infections of the nasal cavity and eye areas, detected at birth in 4 lambs, were resolved within 8 days post-partum. The only pathological effects detected that could possibly be attributed to ureaplasma infection were a placentitis in an ewe that aborted, and the resorption of the foetus in another.

Variable persistence of the contagious equine metritis organism in the genital tract of [formula omitted], [formula omitted], [formula omitted], [formula omitted] and congenitally thymus-deficient (nude) mice

Five inbred strains of mice, CBA J , CBA N , LAF 1, BALB c and congenitally thymus-deficient nude mice of BALB c background, varied considerably in their susceptibility to the contagious equine metritis organism (CEMO). Whereas all the strains were virtually refractive to vaginal challenge, LAF 1 and CBA N mice were readily infected by intra-uterine inoculation. Based on infection rate and nature of the bacteriological response, CBA N mice appeared the more susceptible of the 2 strains. Attempts to transmit CEMO to thymus-deficient nude mice were unsuccessful by both of these routes of challenge and by intraperitoneal inoculation, indicating that host resistance to the causal agent is independent of thymus-mediated immune phenomena. No clinical evidence of infection was observed in any of the experimentally infected mice. Although persistence of CEMO in the female reproductive tract varied widely, it could be isolated from some of the CBA N mice for as long as 19 weeks after challenge by the intra-uterine route. The organism was cultured from the ovaries and/or oviducts of a high percentage of one group of CBA N mice after 50 days, when it could no longer be recovered from the remainder of the genital tract. Limited attempts to achieve venereal transmission of CEMO between culture-positive female and companion male CBA N mice were unsuccessful. The relative susceptibility of the CBA N strain of mice to CEMO would suggest that host resistance to this infection is at least partly dependent on the presence of a fully functional B lymphocyte system. Further studies in this experimental model may elucidate some of the immunological mechanisms underlying development of resistance in the horse, more specifically as they relate to the occurrence of the carrier state in this disease.

Intrauterine inoculation of a bacteria-free filtrate of streptococcus zooepidemicus in clinically normal and infected mares

The effects of the intrauterine inoculation of a bacteria-free filtrate of Streptcoccus zooepidemicus (Strep, filtrate) were studied in a group of mares with clinically normal and chronically inflamed uteri. The reaction was monitored at predetermined intervals by clinical, histological and bacteriological examination. The response was dramatic, although transient, and characterized by an acute suppurative endometritis in all test mares. The control group, infused with Brain-Heart Infusion Broth (BHIB) exhibited a less severe reaction which consisted primarily of an acute eosinophilic endometritis. The Strep filtrate inoculation did not interfere with the subsequent ability of the mares to conceive and carry a foal to term, and has proven to be a useful therapy in cases where previous antibiotic treatments have failed to rid the mare of a chronic infection.

Molecular epidemiology and pathogenesis of some equine herpesvirus type 1 (equine abortion virus) and type 4 (equine rhinopneumonitis virus) isolates.

Representative strains of EHV isolated from an aborted foetus and from a horse with rhinopneumonitis in New Zealand had restriction endonuclease DNA fingerprints typical of those usually associated with these syndromes elsewhere and now designated EHV1 and 4 respectively. EHV1 was isolated from the brain and spinal cord of a 4-year-old gelding that died of myeloencephalitis. A mare on the same farm, at about the same time as the gelding developed myeloencephalitis, aborted and EHV1 was isolated from the tissues of the aborted foetus. Restriction endonuclease DNA fingerprints of the viruses isolated from myeloencephalitis and abortion were indistinguishable by Bam HI but were distinguishable using Bgl I, Pvu II, Xho I and Hind III. The restriction endonuclease DNA fingerprints of 3 EHV1 strains known to cause myeloencephalitis were compared with each other and with EHV1 strains not known to be associated with myeloencephalitis. The Bgl I Pvu II and Hind III DNA fingerprints of the 3 myeloencephalogenic strains appear distinguishable from non-myeloencephalogenic strains. Abortion was induced in a mare by intrauterine inoculation of EHV4. The Bam HI, Bgl I, Pvu II, Xho I and Hind III restriction endonuclease DNA fingerprints of the inoculum virus were indistinguishable from virus recovered from the foetus. It was concluded that passage of the virus through the foetus did not detectably alter the restriction endonuclease DNA fingerprint.

Pseudorabies virus infection of six- and ten-day-old porcine embryos

Nine susceptible gilts were exposed to pseudorabies virus (PrV) by intrauterine inoculation immediately after breeding. Embryos were collected from each of three gilts on days 3, 6, and 10 following exposure to PrV. The number of embryos collected from each gilt was compared with the number of corpora lutea (CL). On days 6 and 10, there were substantially fewer embryos collected than there were CL. The embryos were examined for the presence of viral particles by electron microscopy. PrV was observed in embryos collected at 6 and 10 days following exposure of the gilts. The fluids used to flush the embryos from the uterus during collection were tested for PrV by virus isolation and direct fluorescent antibody procedures. PrV was isolated from the uterine-flush fluids of one of three gilts at each time of embryo collection.

Specific cross-protective antigonococcal immunity in the murine genital tract.

Specific acquired immunity to gonococci was studied in systemically immunized mice, challenged with 10(7) gonococci by intrauterine inoculation. Protection after intraperitoneal immunization was monitored by vaginal cultures taken 24 h post-challenge, since events during the first 24 h postexposure to gonococci are crucial in determining the outcome of infection. Mice were protected against gonococcal challenge by two inoculations with either live or boiled gonococci given 4 weeks apart, whereas immunization with one inoculation did not protect against challenge 1 week later. Protection was correlated with high titers of IgG antibody in serum after two immunizations, but not with the high titers of serum IgM antibody found after the one immunization. IgG antibodies, but not IgM antibodies, were shown to pass into genital secretions. Protection could be passively transferred by serum with high titers of antibody. Of most practical importance was the finding that not only were heat-stable antigens protective, but also heterologous protection resulted after immunization with three strains differing in source (disseminated gonococcal infection versus gonorrhea), opacity-transparency characteristics, and serum sensitivity. The data indicate that IgG antibodies resulting from systemic immunization with heat-stable antigens may be able to provide cross-protection immunity against gonorrhea.

Infertility as a Consequence of Chlamydial Infection of the Upper Genital Tract in Female Mice

The effect of infection with Chalmydia trachomatis (mouse pneumonitis biovar) on the fertility of female mice was examined. Mice were inoculated into one ovarian bursa with approximately 10(4) inclusion-forming units of C. trachomatis and were mated five to 16 days later. At sacrifice all mice showed salpingitis or hydrosalpinx in the inoculated oviduct and implantations were found only in the uterine horn contiguous with the noninoculated oviduct. Animals inoculated with tissue culture supernate showed no tubal pathology and had implantations evenly distributed in both uterine horns. Intrauterine inoculation of Chlamydia five to 36 days before mating also resulted in tubal pathology and infertility. Intrauterine inoculation of C. trachomatis one to two days after mating (one to two days before expected nidation) had no effect on the number or distribution of implantation sites as compared with controls. These results demonstrate that chlamydial infection of the upper reproductive tract of mice prior to mating can result in infertility.

Effects of intrauterine challenge with [formula omitted][formula omitted] serovar [formula omitted] on fertility in cattle

The purpose of this study was to determine the effects of Leptospira interrogans serovar hardio on fertility in cattle. Twenty seronegative mature dairy cows were assigned to two groups. Group I (challenged cows) was bred by a seronegative bull followed by intrauterine infusion (within 30 minutes) of Leptospira interrogans serovar hardjo . Group II was bred by the same bull followed by intrauterine infusion of 5 ml of sterile culture medium. Blood samples were collected at two-day intervals to monitor serum antibody titers. Daily blood cultures for 10 days and weekly urine cultures for five weeks were performed to monitor the animals for leptospiremia and leptospiuria. Cows were slaughtered 35 days post-breeding, and their reproductive tracts were examined. All animals remained clinically normal following intrauterine challenge. There was no difference in pregnancy rates (Group I, 7 10 ; Group II, 6 10 ). All embryos, reproductive tracts, and kidneys appeared normal. A microscopic agglutination test (MA) showed that 4 of 10 challenged cows developed serum antibody titers between 8 and 20 days after challenge. However, on the basis of the hamster passive protection test, all challenged cows had serum antibodies present. All blood and urine cultures were negative through the experimental period, as were the final kidney and uterine cultures. In a second experiment, six seronegative cows were infused with killed microorganisms immediately after insemination. Results of a microscopic agglutination test and a hamster passive protection test indicated that these cows did not develop humoral antibodies against serovar hardjo . These results indicated that intrauterine inoculation of Leptospira interrogans serovar hardjo (hamster-adapted strain) following breeding did not affect pregnancy rates despite an intrauterine challenge which caused the development of humoral antibodies.

Lesions of contagious equine metritis in mares.

Twenty-three mares were infected with contagious equine metritis organism by intrauterine inoculation, and necropsied after intervals of two to 116 days. Severe diffuse subacute salpingitis was seen in one mare, and mild multifocal subacute salpingitis was common. Severe diffuse endometritis and cervicitis initially were acute and became more severe, subacute and predominantly plasmacytic by 14 days, then declined but persisted as mild diffuse or multifocal inflammation for the rest of the experimental period. Vaginitis arose in parallel but resolved after 70 days. There were no lesions in the clitoral fossa or clitoral sinus.

An attempt to isolate [formula omitted][formula omitted] from uterine flushings of brucellosis-reactor donor cattle

Two experiments were conducted to test for the recovery of brucella organisms from uterine flushings and harvested embryos of sero-positive embryo donor females. In Experiment I, 16 sero-positive cows were superovulated with FSH treatments and artificially inseminated at 12, 24 and 36 hours following the onset of estrus with brucella-free semen. At 48 hours after the onset of estrus, one half the potential donor females were administered an intrauterine inoculation of 3.3 to 4.6 × 10 4 Brucella abortus (strain 2308) organisms while the remainder received a control inoculation. In Experiment II, the same 16 cows were similarly administered superovulatory treatments and inseminated following estrus. The uterine inoculation was increased to 1.5 to 2.5 × 10 8 organisms administered 48 hours following estrus. Samples of recovered flushing medium and homogenized embryo residues were placed into a validated in vitro culture system to detect the presence of brucella bacteria. Uterine flushings and embryos recovered from 31 females exhibiting estrus following FSH treatments were free from either field strain or the inoculated B. abortus (strain 2308) contamination. The flushings obtained from a single female, which did not respond with estrus following FSH treatment but was inoculated at appointment, did contain B. abortus which was identified as the inoculated strain 2308 and not field strain organisms. These results indicate that brucella contamination of flushing media and harvested embryos will not likely be incurred when collecting embryos from sero-positive donor females. These findings offer further encouragement for the use of embryo transplantation as a method to produce brucella-free offspring from infected cows.

Contagious equine metritis: effect of intrauterine inoculation of tiny colony forms in pony mares.

Contagious equine metritis: effect of intrauterine inoculation of tiny colony forms in pony mares.