Effects of re-infection with Neospora caninum in bulls on parasite detection in semen and blood and immunological responses

Three bulls with experimentally induced primary infection with Neospora caninum were re-infected intravenously with 10(8) live N. caninum tachyzoites of the NC-1 isolate at 300 days post-infection to investigate the presence of N. caninum in semen and blood, and the associated immune responses. In parallel, three bulls with experimentally induced primary infection with N. caninum and three non-infected bulls were also monitored. Re-infected and infected bulls showed an intermittent presence of N. caninum DNA in semen with a parasite load ranging from 0.1 to 15.6 (mean 4.4) and 0.1 to 11.1 (mean 4.1) parasites/ml, respectively. Re-infected bulls showed significant and persistent serum-specific IgM and IgG antibody responses. Specific IgG levels were detected in seminal plasma of all infected bulls, but the magnitude of the response was significantly higher in re-infected rather than in chronically infected animals. The mean specific IFN-gamma levels in re-infected bulls were significantly increased as early as 3 and 7 days after experimental infection when compared to bulls in other groups. This study showed that the intermittent presence and parasite load of N. caninum in the semen of re-infected bulls is very similar to that reported in chronically infected animals. The protozoa could not be isolated from BALB/c nu/nu mice inoculated with PCR-positive semen samples and inseminated heifers with pooled semen samples did not show seroconversion. Plasma IFN-gamma level seems to be a good indicator of a recent N. caninum infection.     

Fertility in heifers and cows after low dose insemination with sex-sorted and non-sorted sperm under field conditions

The present study was performed to test fertility after low dose insemination with sexed and non-sexed sperm in dairy cattle under field conditions in Switzerland. Spermatozoa were stained with Hoechst 33342 and sorted by flow cytometry. A total of 132 heifers and cows were inseminated with 2 x 10(6) X-bearing, frozen-thawed sperm (A) and 91 animals were inseminated with the same dose using non-sorted, frozen-thawed sperm (B). Pregnancy examination by ultrasound was performed twice, 30-40 days (PE1) and 70-90 days (PE2) after insemination. The pregnancy rates after PE1 were 33.3% (9/27) and 59.3% (16/27) in heifers (P=0.05) and 27.6% (29/105) and 28.1% (18/64) in cows (P>0.05) for groups A and B, respectively. Embryonic losses between PE1 and PE2 in heifers were 11.1% (1/9) and 0% (0/16) and in cows 17.2% (5/29) and 5.6% (1/18), the differences between groups A and B not being significant (P>0.05). Calving rates in heifers were 29.6% (8/27) and 57.8% (15/26), whereas in cows 22.1% (23/104) and 23.4% (16/63) gave birth to calves (for both groups P>0.05). The sex ratio was different (P<0.05) between A (85.3%) and B (58.6%). From our results it can be concluded that conception rates of sorted and non-sorted semen are similar using an insemination dose of 2 x 10(6). Fertility may be increased by improving sexing technology and animal management.     

Experimental neosporosis in bulls: parasite detection in semen and blood and specific antibody and interferon-gamma responses

AIM: To investigate the presence of Neospora caninum in semen and blood, and the development of specific antibody and interferon-gamma (IFN-gamma) responses in experimentally infected bulls. METHODS: Eight bulls were intravenously infected with 10(8) live N. caninum tachyzoites of NC-1 isolate. The presence of N. caninum in semen and blood was assessed using a nested-PCR procedure. PCR-positive semen samples were bioassayed using a BALB/c nu/nu mouse model. Specific anti-N. caninum antibody and IFN-gamma responses were also examined. In parallel, eight seronegative bulls were studied as non-infected controls. All bulls were monitored for 26 weeks. RESULTS: All eight experimentally infected bulls showed N. caninum DNA in their semen and/or blood samples at some time during the course of the study. Parasite load in semen ranged from 0.1 to 14.5 parasites/ml (mean 6.0). N. caninum could not be detected in BALB/c nu/nu mice inoculated with PCR-positive semen samples. A significant increase in mean serum specific IgM antibody response to N. caninum was detected between 10 and 28 days post-infection (p.i.). Serum specific IgG, IgG1, and IgG2 antibody levels in experimentally infected bulls were significantly different after 21, 10, and 14 days p.i. as compared to controls, respectively. Specific anti-N. caninum IgG were detected in seminal plasma from infected bulls and values obtained were different from controls after 25 days p.i. Mean specific IFN-gamma responses in experimentally infected bulls were significantly higher than controls 3 days p.i. CONCLUSIONS: This is the first study to report the presence of N. caninum DNA in the semen and blood of experimentally infected bulls. Our observations indicate an intermittent presence of N. caninum in low numbers in semen and associated with chronic stage of the infection. This study is also the first to report the detection of anti-N. caninum IgG in seminal plasma of experimentally infected bulls.     

Embryo production from superovulated Holstein-Friesian dairy heifers and cows after insemination with frozen-thawed sex-sorted X spermatozoa or unsorted semen

The aim of this study was to evaluate embryo production in superovulated Holstein-Friesian dairy heifers and cows inseminated with either X-sorted spermatozoa (2 million/dose) or unsorted semen (15 million/dose). Experiment 1 at the research farm involved eight heifers, six cows and semen of one Holstein bull. All transferable embryos were diagnosed for sex. Experiment 2 included embryo collections on commercial dairy farms: X-sorted spermatozoa from three Holstein bulls were used for 59 collections on 28 farms and unsorted semen from 32 Holstein bulls were used for 179 collections on 79 farms. Superovulations were induced by eight declining doses of FSH (total of 12 ml for heifers and 19 ml for cows) starting on days 8-12 of the estrus cycle. Inseminations began 12h after the onset of estrus and were performed two to four times at 9-15 h intervals. Low-dose X-sorted inseminates were deposited into uterine horns and unsorted semen was placed into the uterine body. In Experiment 1, on average 70.3 and 75.0% of embryos recovered from heifers, and 48.4 and 100% of embryos recovered from cows were of transferable quality in X-sorted and unsorted groups, respectively. The proportion of transferable female embryos produced approximately doubled when insemination was with X-sorted spermatozoa compared to insemination with unsorted semen (heifers 96.4% versus 41.1%; cows 81.1% versus 39.8%). In Experiment 2, estimated 53.9 and 65.5% of embryos recovered from heifers, and 21.1 and 64.5% of embryos recovered from cows were of transferable quality in X-sorted and unsorted groups, respectively. Proportions of unfertilized oocytes were 21.1 and 10.6% for heifers and 56.0 and 14.4% for cows in X-sorted and unsorted groups, respectively. Consequently, cows inseminated with X-sorted spermatozoa produced significantly smaller proportions of transferable embryos (p<0.005) and significantly larger proportions of unfertilized oocytes (p<0.001) than those inseminated with unsorted semen. Proportions of quality 1 or degenerated embryos were similar for the two treatments in both heifers and cows. Within treatments, bulls did not significantly affect the proportions of transferable, unfertilized or degenerated oocytes/embryos. It was concluded that using low-dose X-sorted spermatozoa rather than normal-dose unsorted semen for the insemination of superovulated embryo donors can improve the proportion of transferable female embryos produced but this potential may not be achieved in commercial practice, particularly in cows, because of reduced fertilization rates when using low doses of X-sorted spermatozoa.     

Embryo production with sex-sorted semen in superovulated dairy heifers and cows

The aim of this study was to examine the effect of sex-sorted semen on the number and quality of embryos recovered from superovulated heifers and cows on commercial dairy farm conditions in Finland. The data consist of 1487 commercial embryo collections performed on 633 and 854 animals of Holstein and Finnish Ayrshire breeds, respectively. Superovulation was induced by eight intramuscular injections of follicle-stimulating hormone, at 12-hour intervals over 4 days, involving declining doses beginning on 9 to 12 days after the onset of standing estrus. The donors were inseminated at 9 to 15–hour intervals beginning 12 hours after the onset of estrus with 2 + 2 (+1) doses of sex-sorted frozen-thawed semen (N = 218) into the uterine horns or with 1 + 1 (+1) doses of conventional frozen-thawed semen (N = 1269) into the uterine corpus. Most conventional semen (222 bulls) straws contained 15 million sperm (total number 30–45 million per donor). Sex-sorted semen (61 bulls) straws contained 2 million sperm (total number 8–14 million per donor). Mean number of transferable embryos in recoveries from cows bred with sex-sorted semen was 4.9, which is significantly lower than 9.1 transferable embryos recovered when using conventional semen (P ≤ 0.001). In heifers, no significant difference was detected between mean number of transferable embryos in recoveries using sex-sorted semen and conventional semen (6.1 and 7.2, respectively). The number of unfertilized ova was higher when using sex-sorted semen than when using conventional semen in heifers (P < 0.01) and in cows (P < 0.05), and the number of degenerated embryos in cows (P < 0.01), but not in heifers. It was concluded that the insemination protocol used seemed to be adequate for heifers. In superovulated cows, an optimal protocol for using sex-sorted semen remains to be found.     

Susceptibility of Psammomys obesus and Meriones tristrami to tachyzoites of Neospora caninum

The susceptibility of Psammomys obesus (sand rat) and Meriones tristrami (Tristram's jird) to Neospora caninum was investigated by subcutaneous (s.c.) and intraperitoneal (i.p.) inoculation of 10-fold doses of culture-derived tachyzoites. Groups of 5 animals were inoculated with doses of 10-10(7) parasites via each route of inoculation. All but 2 of the sand rats inoculated with doses of 10-10(4) parasites succumbed to the infection by 7-18 days postinfection. All jirds inoculated with 10(7) tachyzoites succumbed by 5-16 days postinfection and those inoculated with 10(6) tachyzoites by 9-25 days. A considerable proportion of the jirds inoculated with 10-10(5) tachyzoites survived. Fibrinous peritonitis with ascites containing numerous tachyzoites was observed in the i.p.-inoculated sand rats and jirds that succumbed to the infection. In the jirds, tachyzoites were also found in pleural exudate. A considerable number (42.8%) of the jirds inoculated s.c. or i.p. exhibited neuromuscular symptoms, expressed in ataxia, head tilt, circling movement, and posterior paralysis. Seven successive passage of tachyzoites were achieved in sand rats with doses of 10(5) parasites and in jirds with doses of 10(7) parasites. All surviving jirds became seroconverted and were immune to lethal challenge.     

Growth of and competition between Neospora caninum and Toxoplasma gondii in vitro

Competitive interactions between Neospora caninum and Toxoplasma gondii were studied because both species appear to have identical ecological niches in vitro. Tachyzoites of N. caninum (NC-1 isolate) and T. gondii (RH isolate) were compared in three in vitro studies: (1) rate of penetration of host cells; (2) generation time; and (3) competition between the two species when grown together in the same flask and allowed to compete for space. When tachyzoites of the two species were inoculated onto human foreskin fibroblasts, 3.24-times more N. caninum tachyzoites penetrated cells by 1 h p.i. At 3 h p.i., there were 2.87-times more N. caninum intracellular tachyzoites than T. gondii tachyzoites. The generation times for N. caninum (NC-1 isolate) and T. gondii (RH isolate) were approximately 14-15 h and 8-10 h, respectively. Before exponential growth occurred, both species displayed a lag period, which was 10-12 h for N. caninum and 8-10 h for T. gondii. To observe competition, equal numbers of tachyzoites of each species were mixed and inoculated into flasks of host cells, and the monolayers were allowed to proceed to >90% lysis before the next transfer. Competition was analysed for 31 days by labelling samples of each flask with a species-specific monoclonal antibody and determining the ratio of each species. In all trials, T. gondii outcompeted N. caninum. By 4 days p.i., 70% of the tachyzoites were T. gondii; this percentage increased to 97% by 23 days p.i. When the starting inoculum contained 75% N. caninum and 25% T. gondii tachyzoites, T. gondii was still competitively superior. When infected monolayers that were labelled with T. gondii-specific antibodies were examined, it was noted that both species can occupy and undergo endodyogeny in the same host simultaneously.     

A POLYGEN-adjuvanted killed Neospora caninum tachyzoite preparation failed to prevent foetal infection in pregnant cattle following i.v./i.m. experimental tachyzoite challenge

Cattle immunised with a POLYGEN-adjuvanted killed Neospora caninum tachyzoite preparation were previously shown to produce interferon (IFN)-gamma at levels similar to those of tachyzoite-infected cattle. In view of the critical role of IFN-gamma in resistance of mice to N. caninum infection, these results prompted us to test the POLYGEN-adjuvanted preparation in pregnant cattle to determine whether it will be able to prevent foetal infection following an experimental tachyzoite challenge. Seven heifers were immunised at 35 and 63 days of gestation with the POLYGEN-adjuvanted preparation, while five heifers were inoculated with POLYGEN alone at the same days of gestation. Four weeks later, all heifers were challenged with a combined i.v./i.m. inoculation of tachyzoites. The same challenge was given to seven unimmunized heifers at the same stage of gestation. An additional unimmunized heifer was inoculated with uninfected monolayer cell culture material. All challenged heifers, immunized and unimmunized, had infected foetuses. Immunized heifers developed both parasite-specific humoral and cellular immune responses, characterised by increased IFAT titres, a predominant IgG1 response, elevated lymphoproliferative response and IFN-gamma production. Following tachyzoite challenge, they developed an anamnestic humoral response and produced similar amounts of IgG1 and IgG2 antibodies, but did not have an anamnestic cellular immune response. The lack of anamnestic cellular immune response and/or the large i.v/i.m tachyzoite inoculum may have contributed to the failure of the preparation.     

Induction of ovulatory estrus and fertility in non-cycling buffaloes with norgestomet during low breeding season

Field trials were designed to evaluate use of norgestomet treatment to induce ovulatory estrus in non-cycling buffalo cows and heifers during low breeding season. Twenty-five buffalo cows and 50 heifers under village management were given a 9-day treatment with a polymer implant containing 6 mg norgestomet with IM injections of 5 mg estradiol valerate + 3 mg norgestomet at the time of implantation and 600 IU PMSG when the implant was removed. Fifty animals served as controls without any treatment. Seventy-four treated animals showed estrus during the period between 36 to 80 hours after removal of the implant. Twenty-five buffalo cows and 40 heifers that could be further followed up were inseminated twice at 8-hour intervals, 12 hrs after induction of estrus with chilled semen by recto-vaginal method. Of these, 15 (23.1%) conceived, 9 (36%) among buffalo cows and 6 (15%) among heifers. Fourteen buffalo cows and 30 heifers that did not conceive manifested cyclic estrus at an interval of 22.4 and 20.6 days. The conception rate in the cyclic estrus was 57% and 23.3%, respectively, for buffalo cows and heifers. The overall conception rate over two inseminations was 46.2%, 68% in buffalo cows and 32.5% in heifers. In the control group, five (10%) showed spontaneous estrus and two (40%) conceived during the period of the experiment.     

Effect of widespread and limited use of sexed semen on genetic progress and reproductive performance of dairy cows

Stochastic simulation was used for studying the impacts of sexed semen on genetic progress and reproductive performance of dairy cows. Three strategies were compared: WSS (use unsexed semen in cows and heifers), SSH (use sexed semen in heifers and unsexed semen in cows) and SSCH (use sexed semen in both cows and heifers). Conception rate (CR) of unsexed semen was considered to be 35% and 65% in cows and heifers, respectively. CR of sexed semen was considered to be 15 (20% in cows and 50% in heifers), 10, 5 and 0 percentage points lower than unsexed semen. Thus, four subschemes were compared under SSCH (SSCH15, SSCH10, SSCH5, SSCH0) and SSH (SSH15, SSH10, SSH5, SSH0). Moreover, the effect was studied in four distinct paths of selection: active sires (AS), young bulls (YB), bull dams (BD) and milking cows (CW). The average genetic superiority of CW was 12% and 9.5% in SSCH15 and SSH15 strategies relative to a base scheme, respectively. The average genetic superiority of CW was 19% and 10.5% in SSCH0 and SSH0, respectively. Regression analysis showed that genetic superiority of CW increased significantly, that is, 0.5% and 0.1% per every 1% increase in CR in SSCH and SSH, respectively. The result showed that there is a significant difference between genetic superiority of cows in SSCH and SSH schemes. Widespread and limited use of sexed semen in commercial dairy herds resulted in a large genetic advantage in CW. The genetic advantage of gender control was minimal in the selection paths of AS, YB and BD. Open days and services per conception reached to 153 v. 125 days and 5 v. 2.86 under SSCH15 compared with WSS. The age at first calving increased from 774 to 790 days in SSH15 and SSCH15 strategies. Mean of parities decreased to 2.26 v. 2.42 by using sexed semen. The widespread use of sexed semen increased the age average of cows in all parities. Sexed semen increased selection intensity in the CW path, and this contributed to the genetic merit of future cows. On the other hand, sexed semen had a negative effect on the reproductive performance of dairy cows. Generally, although the effect of widespread use of sexed semen (SSCH) on genetic progress is significantly more than limited use of sexed semen (SSH), SSCH decreased reproductive performance of dairy herds dramatically, and this suggests that SSH scenarios might be more appropriate in animal breeding programs. Finally, to make a decision of which schemes are more convenient, it is necessary to compare the economic aspects of schemes.     

Semen variables of sheep (Ovis aries) experimentally infected with Toxoplasma gondii

The influence of Toxoplasma gondii on semen variables and sperm morphology of sheep was evaluated in eight reproductive males distributed into three experimental groups: GI, three sheep inoculated with 2.0x10(5) of P strain oocytes; GII, three sheep infected with 1.0x10(6) of RH strain tachyzoites and; GIII two control sheep. Clinical (rectal temperature, cardiac and respiratory frequencies), parasite and serology exams (IIF) were realized. Sperm variables (volume, motility, vigor and concentration) and semen morphology for each sheep were also evaluated. Thus, semen and blood collections were assessed on post-inoculation days (PIDs)-1,3,5,7,11,14 and weekly thereafter up to PID 70. Clinical alterations were observed (hypothermia and anorexia) in infected sheep from groups GI and GII. Parasitic outbreaks were detected in five sheep. All the infected sheep produced antibodies against T. gondii from PID 5 onwards, reaching a peak of 4096 and 8192 for group GI and GII sheep, respectively. Differences (P<0.05) were observed regarding the ejaculate volume between the inoculated groups (oocytes and tachyzoites) and control. Even though experimental toxoplasmic infection resulted in clinical symptomology in the inoculated sheep, the minimal alterations in sperm pathologies could not be directly attributed to T. gondii.     

Effect of sperm number and frequency of insemination on fertility of mares inseminated with cooled semen

In this study, we tested the hypothesis that insemination of mares with twice the recommended dose of cooled semen (2 x 10(9) spermatozoa) would result in higher pregnancy rates than insemination with a single dose (1 x 10(9) spermatozoa) or with 1 x 10(9) spermatozoa on each of 2 consecutive days. A total of 83 cycles from 61 mares was used. Mares were randomly assigned to 1 of 3 treatment groups when a 40-mm follicle was detected by palpation and ultrasonography. Mares in Group 1 were inseminated with 1 x 10(9) progressively motile spermatozoa that had been cooled in a passive cooling unit to 5 degrees C and stored for 24 h. A second aliquot of semen from the same collection was stored for an additional 24 h and inseminated at 48 h after collection. Mares in Group 2 were inseminated once with 1 x 10(9) progressively motile spermatozoa that had been cooled to 5 degrees C and stored for 24 h. Group 3 mares were inseminated once with 2 x 10(9) progressively motile spermatozoa that had been cooled to 5 degrees C and stored for 24 h. All mares were given 2500 IU i.v. hCG at the first insemination. Pregnancy was determined by ultrasonography 12, 14 and 16 d after ovulation. On Day 16, mares were administered i.m. 10 mg of PGF2 alpha and, upon returning to estrus, were randomly reassigned to a group for repeated treatment. Semen was collected from one of 3 stallions every 3 d; mares with a 40-mm ovarian follicle were inseminated with semen from the stallion collected on the preceding day. Semen was allocated into doses containing 1 x 10(9) progressively motile spermatozoa, diluted with dried skim milk-glucose extender to a concentration of 25 x 10(6) motile spermatozoa/ml (total volume 40 ml), placed in a passive cooling unit and cooled to 5 degrees C for 24 or 48 h. Response was measured by number of mares showing pregnancy. Data were analyzed by Chi square. Mares inseminated twice with 1 x 10(9) progressively motile spermatozoa on each of two consecutive days had a higher pregnancy rate (16/25, 64%; P < 0.05) than mares inseminated once with 1 x 10(9) progressively motile spermatozoa (9/29, 31%) or those inseminated once with 2 x 10(9) progressively motile spermatozoa (12/29, 41%). Pregnancy rates did not differ significantly (P > 0.10) among stallions (69, 34 and 32%). Interval from last insemination to ovulation was 0.9, 2.0 and 2.0 d for mares in Groups 1, 2 and 3, respectively. Based on these results, the optimal insemination regimen is a dose of 1 x 10(9) progressively motile spermatozoa given on two consecutive days. However, a shorter interval (< or = 24 h rather than > 0.9 d) between insemination and ovulation may affect pregnancy rates, and needs to be investigated.     

The effect of a diet containing excess quickly degradable nitrogen (QDN) on reproductive and metabolic hormonal profiles of lactating dairy cows

The objective of this experiment was to examine the effects of an excess intake of quickly degradable nitrogen (QDN) on metabolic and reproductive parameters in lactating dairy cows. Twenty-two lactating dairy cows were fed a total mixed ration once daily. The control diet was a typical ration for high producing cows in the UK (CP = 17.5%, ME = 11.8 MJ/kg DM). The cows were randomly divided into two groups, control diet (control; n = 12) and excess QDN diet (QDN; n = 10). The QDN group was fed an additional 250 g of urea per cow per day, from 10 days before insemination (day 0) until the end of the experiment, 17 days after the second insemination. Ten days before insemination, a synchronized oestrus was induced and the cows inseminated twice, 48 and 72 h after synchronization, with commercial frozen semen from a single sire. Ovaries were scanned using B-mode ultrasonography 10 days before insemination and then daily from 3 days before insemination. Eighteen of the cows (9 control and 9 QDN) were sampled more intensively to determine the pulsatile pattern of secretion of luteinizing hormone (LH) and growth hormone (GH). Cows were slaughtered 17 days after insemination, the reproductive tracts recovered and flushed to retrieve embryos. The excess QDN diet resulted in elevated (P < 0.05) plasma urea concentrations 3 days after starting urea feeding and these were maintained until the end of the experiment. However, the excess QDN diet did not significantly affect daily milk production or plasma concentrations of insulin and IGF-I. The QDN treatment did not significantly affect pulsatile patterns of secretion of LH and GH or the number of small (< 0.5 cm diameter) and medium to large follicles (> 0.5 cm diameter). Twenty cows ovulated following synchronization (control 11/12; QDN 9/10). There were no significant differences between the control and the QDN groups in the peak concentrations of oestradiol during the follicular phase or in the post-ovulatory pattern of plasma and milk progesterone secretion. Embryos and/or foetal membranes were recovered from 10 cows (5 control and 5 QDN). The results of the current study show that feeding excess QDN, as urea, for 27 days commencing 10 days before insemination had no effect on reproductive or metabolic hormonal parameters. Ovulation and the formation and function of the post-ovulatory corpus luteum were also unaffected by excess QDN. These data suggest that the harmful effects of excess intakes of QDN on fertility occur after 17 days following ovulation.     

Transplacental Neospora caninum infection in cats

Transplacental transmission of Neospora caninum was studied in 2 pregnant cats (queens). Queen 1 was inoculated subcutaneously with 2 x 10(6) cell culture-derived N. caninum tachyzoites on day 47 of gestation. She gave birth to a full-term kitten on the 17th day after inoculation. The kitten died the second day after birth due to generalized N. caninum infection. The mother cat was killed on the third day after parturition and was found to have a macerated kitten in the uterus. Severe placentitis, metritis, hepatitis, and nephritis due to N. caninum were seen in tissues from the queen. Queen 2 was fed N. caninum tissue cysts and mated 111 days later. She gave birth to 3 healthy full-term kittens. The kittens were necropsied at 2, 22, and 30 days of age. Neospora caninum was recovered from the organs and was seen in histologic sections in 1 of the 3 kittens. Results indicate that N. caninum can be transplacentally transmitted in cats during acute and chronic stages of infection. Neospora caninum-specific IgG antibodies were demonstrated in the sera of inoculated cats and nursing kittens.     

Immunisation of mice against neosporosis

In the present study a murine encephalitis model was used to investigate if protection against neosporosis could be achieved by immunisation. Groups of 10 mice were immunised with a sublethal dose of live Neospora caninum tachyzoites, N. caninum antigens incorporated into iscoms, N. caninum lysate mixed with Quil A, or N. caninum lysate in PBS. Control mice were given Quil A only. Challenge infection with 2.5x10(6) N. caninum tachyzoites resulted in clinical symptoms that remained until the end of the experiment in the controls. In contrast, mice immunised with live parasites or parasite lysate in Quil A only showed mild and transient symptoms. Of nine mice immunised with N. caninum iscoms, seven recovered while two died. Most severely affected were the mice immunised with parasite lysate only; all of them died within 28 days post-infection. Histological examination and scoring of brain lesions gave a significantly lower (P<0.0001) lesion score in mice immunised with live parasites than in controls. The groups immunised with iscoms or lysate and Quil A also had reduced lesion scores (P<0.04 and 0.07, respectively) but not the group given parasite lysate alone. The lesions seen in the latter group differed from those in the other groups. There was less cellular reaction and more tachyzoites indicating an active infection. The N. caninum specific antibody responses and cytokine production (IFN-gamma, IL-4 and IL-5) of splenocytes were analysed at the time of challenge infection. The results suggest a correlation between protection and high levels of IFN-gamma. Also, the immune responses recorded in mice immunised with parasite lysate without adjuvant were relatively weak and more towards the Th2 type, when compared with the other immunisation schedules. This is consistent with the weaker inflammatory response observed in the brains of these mice.     

The increase in plasma C19Delta5 steroids in subcutaneous abdominal and jugular veins of dairy cattle during pregnancy is unrelated to estrogenic activity

Plasma concentrations of dehydroepiandrosterone (DHEA), 5-androstene-3beta,17beta-diol (AED), and 17beta-estradiol (E2) in dairy cows and heifers and AED binding to uterine cytosolic estrogen receptor (ER) were studied. Plasma samples were collected from the subcutaneous abdominal (SA) and jugular (J) veins of heifers and cows in the non-pregnant state and at 15-45, 90-120, 180-210, and 250-280 days of pregnancy (N = 5-12). Plasma DHEA, AED, and E2 were determined by RIA. DHEA and AED significantly increased (P < 0.001) in heifers and cows throughout pregnancy. The stage of pregnancy significantly (P < 0.001) affected the three steroids in heifers and cows. Plasma DHEA increased throughout pregnancy in both heifers and cows, and in heifers it was significantly greater in SA than in J veins at 90-120 days (P < 0.01). Plasma AED was greater in heifers than in cows in J veins at 90-120 days (P < 0.01) and 180-210 days (P < 0.05), and in SA veins, at 15-45 days (P < 0.01) and 90-120 days (P < 0.05). In heifers, circulating AED showed concentration values significantly greater than those in non-pregnant animals from 90 to 120 days (P < 0.05) and was significantly greater in SA than in J veins at 90-120 days (P < 0.05). In cows, plasma AED was significantly greater than in non-pregnant animals at 250-280 days (P < 0.01). In heifers, plasma E2 was significantly greater in the SA than in the J veins from 180-210 to 250-280 days (P < 0.01). In cows, differences between E2 plasma concentrations in J and SA veins were observed only at 250-280 days of pregnancy. At 250-280 days, in both animal types plasma E2 was significantly greater than in non-pregnant animals (P < 0.001). We suggest that AED originates primarily from the feto-placental unit, while mammary E2 synthesis near term can affect plasma concentrations. Binding data showed that AED is a weak competitor for cytosolic ER (IC50 range: 1.44 x 10(-5) to 3.71 x 10(-5) M). These results suggest that a direct estrogenic activity for AED is unlikely in dairy cattle, and the physiological role of AED needs to be elucidated.     

Vaccination with gamma-irradiated Neospora caninum tachyzoites protects mice against acute challenge with N. caninum

Neospora caninum, an apicomplexan parasite, is a leading cause of bovine abortions worldwide. The efficacy of gamma-irradiated N. caninum strain NC-1 tachyzoites as a vaccine for neosporosis was assessed in C57BL6 mice. A dose of 528 Gy of gamma irradiation was sufficient to arrest replication but not host cell penetration by tachyzoites. Female C57BL6 mice were vaccinated with two intraperitoneal inoculations of 1 x 10(6) irradiated tachyzoites at 4-wk intervals. When stimulated with N. caninum tachyzoite lysates, splenocytes of vaccinated mice, cultured 5 and 10 wk after vaccination, secreted significant (P<0.05) levels of interferon gamma, interleukin (IL)-10, and small amounts of IL-4. Antibody isotype-specific ELISA of sera from vaccinated mice exhibited both IgG1 and IgG2a isotypes of antibodies. Vaccinated mice were challenged intraperitoneally with 2 x 10(7)N. caninum tachyzoites. All vaccinated mice remained healthy and showed no obvious signs of neosporosis up to the 25th day post-challenge when the study was terminated. All unvaccinated control mice died within 1 wk of infection. Gamma-irradiated N. caninum tachyzoites can serve as an effective, attenuated vaccine for N. caninum.     

Neospora caninum (Protozoa: apicomplexa) infections in mice

Groups of mice were given 0 mg, 4 mg, or 2 mg of methylprednisolone acetate (MPA) 7 days prior to, the day of, and 7 days after subcutaneous inoculation with 0 or 2 x 10(5) tachyzoites of Neospora caninum. Clinical signs of disease were seen only in mice given both MPA and N. caninum tachyzoites. Mice given 4 mg MPA and N. caninum tachyzoites developed severe disseminated neosporosis and most died or were killed when comatose 11-13 days postinoculation (PI). Acute pneumonia, polymyositis, encephalitis, hepatitis, and pancreatitis were the main lesions in these mice. Mice given 2 mg MPA and N. caninum developed mild pneumonia and many mice began showing neurological signs 14 days PI. Neurological signs consisted mainly of pronounced head-tilting and associated impairment of movement. Grossly visible 1-2-mm single or multiple, white areas of discoloration were seen in the brains of many of these mice. Encephalitis, ganglioradiculoneuritis, pneumonia, and polymyositis were the main changes seen in these mice. Tissue cysts of N. caninum were only seen in mice given 2 mg MPA and were first seen 21 days PI. Tissue cysts were 16-34 by 13-29 microns and had a 1.5-3.0-microns-thick cyst wall. Tissue cysts were seen only in the brain. Mice given 4 mg MPA and tachyzoites and host cells that had been frozen for 1 wk did not develop clinical signs of infection, indicating that freezing kills tachyzoites and that viruses or other agents were not involved in the genesis of disease seen in mice given MPA and viable tachyzoites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Syncro-mate B induces estrus in ovariectomized cows and heifers

Eleven ovariectomized Hereford x Simmental cows and 10 ovariectomized crossbred heifers (primarily Angus and Hereford) were given the Syncro-Mate B (SMB) estrous synchronization treatment. The SMB treatment consisted of a 2 ml i.m. injection containing 5 mg of estradiol valerate and 3 mg of norgestomet plus a hydron ear implant containing 6 mg of norgestomet. The ear implant was removed 9 d later. Cows and heifers were considered in estrus only if they stood for mounting by a herdmate or a bull. Observations for estrus were made four or six times each day for 3 d after implant removal. The 21 animals were used in eight trials. Each trial involved 9 or 11 cows or 5 or 10 heifers. Four days to three weeks elapsed between implant removal and implant insertion for the next trial. No ovariectomized cow or heifer was observed in estrus for 21 d before treatment with SMB. In the eight trials, 3 of 9, 7 of 9 and 6 of 11 cows exhibited estrus, whereas 5 of 10, 1 of 5, 3 of 5, 3 of 5 and 5 of 5 heifers exhibited estrus after treatment. When data were pooled, 16 of 29 (55.2%) cows and 17 of 30 (56.7%) heifers exhibited estrus after treatment. Our data indicate that the SMB treatment can induce estrus in cows and heifers, independently of the ovaries.     

Oocyte transfer in mares with intrauterine or intraoviductal insemination using fresh, cooled, and frozen stallion semen

The objectives were to compare embryo development rates after oocyte transfer with: (1) intrauterine or intraoviductal inseminations of fresh semen versus intraoviductal insemination of frozen semen; (2) intraoviductal versus intrauterine inseminations of cooled semen. In Experiment I, oocytes were transferred into the oviduct, and recipients were inseminated into the uterus with 1 x 10(9) fresh spermatozoa, or into the oviduct with 2 x 10(5) fresh or frozen-thawed spermatozoa. In Experiment II, semen was cooled to 5 degrees C before intrauterine insemination with 2 x 10(9) spermatozoa or intraoviductal inseminations of 2 x 10(5) spermatozoa (deposited with the oocytes). In Experiment I, embryo development rates were similar (P>0.05) for intrauterine versus intraoviductal inseminations when fresh semen was used (8/14, 57% and 9/11, 82%, respectively). However, embryo development rates were lower (P<0.05) when frozen spermatozoa were placed within the oviduct (1/12, 8%). In Experiment II, embryo development rates were higher (P<0.05) when cooled semen was used for intrauterine (19/23, 83%) versus intraoviductal (4/16, 25%) inseminations. We concluded that intraoviductal insemination can be successfully performed using fresh spermatozoa. However, the use of cooled and frozen spermatozoa for intraoviductal inseminations was less successful, and needs further investigation.