A Study of Complement-fixation and Serum Agglutination Tests on Vibrio fetus Infected and Immunized Cattle

Complement-fixation (CF) and tube agglutination (TA) tests for demonstration of Vibrio fetus antibodies were conducted on the sera of three groups of ten heifers. One group was vaccinated subcutaneously with a commercial V. fetus var venerealis bacterin and challenged intra-utero, at the external os cervicus one month later; the second was infected only and the third vaccinated only.

The vaccinated cattle developed high CF serum titers, but no such increase was observed in animals infected only. A moderate increase in serum antibody titers was demonstrated by the TA test following either infection or vaccination; although titers observed were not higher than those observed in the sera of some apparently normal uninfected animals. The group receiving both vaccine and challenge was the only one in which significant serum antibody titers were demonstrable by the TA test. The sera of these animals also had significant titers in the CF tests.

The CF and TA tests detected serum antibodies produced by the parenteral inoculation of V. fetus antigen. These two tests were of limited value in detecting serum antibodies from animals with genital V. fetus var venerealis infection, although the formation of local antibodies was demonstrable by the vaginal mucus agglutination test.

     

Urinary concentration of corticoids in ponies with hyperlipoproteinaemia or hyperadrenocorticism

Summary

The urinary corticoid:creatinine (c:c) ratio was determined in ten pony mares suffering from hyperlipoproteinaemia. The mean (± sd) urinary c:c ratio of these ten ponies (47 ± 31 x 10^‐6) was not significantly different from that of twelve pony mares with a pituitary pars intermedia adenoma (31 ± 18 x 10^‐6). The correlation between the urinary concentration of corticoids and plasma total lipids, and the correlation between the urinary c:c ratio and plasma total lipids in ponies with hyperlipoproteinaemia were not significant (P > 0.05; r=0.53 and r=‐0.008, respectively).

Preliminary results favour primary hyperadrenocorticism being associated with hyperlipoproteinaemia. In conclusion, the data presented here suggest that cortisol can contribute to insulin resistance in ponies with hyperlipoproteinaemia.     

The role of serum and biliary antibodies and cell-mediated immunity in the clearance of S. typhimurium from chickens

The development of three parameters of immunity in response to a non-lethal infection of $\text{Salmonella typhimurium}$ in adult chickens has been examined. Intravenous inoculation of 1 × 10⁶ organisms established infection in the liver, spleen and intestinal tract of all birds; the organism persisted in these sites until day 9 of the infection, after which it was cleared rapidly from all sites. High levels of agglutinating and haemagglutinating antibodies were found in serum and bile 5 days after infection; they peaked at days 7 to 10, and detectable antibody was still present in both fluids 6 weeks after infection. The presence of this antibody did not appear to cause a significant reduction in organism numbers in any of the sites examined. Cell mediated immunity was detected at day 14. It is suggested that cell mediated immunity is responsible for clearance of the organisms from the tissues.     

Opsonins of Streptococcus in uterine flushings of mares susceptible and resistant to endometritis: control of secretion and partial characterization

The release of opsonins into the uterine lumen of mares susceptible or resistant to endometritis was examined after intrauterine inoculation of a filtrate of Streptococcus culture fluid or vehicle. Uterine flushings were collected at 0.5 hour before and 2, 4, 6, 8, and 24 hours after inoculation on day 2 or 3 of estrus and on day 7 or 8 after ovulation. Amounts of opsonins in flushings were quantified as the H2O2 produced by leukocytes incubated with flushings-opsonized bacteria, compared with H2O2 produced by leukocytes incubated with nonopsonized bacteria. Opsonin values in flushings increased (P less than 0.025) in all mares after inoculation of filtrate or vehicle. For mares resistant to endometritis, opsonin values were greater at diestrus than at estrus. The opposite was true for mares susceptible to endometritis, resulting in a status (susceptible vs resistant) X stage of cycle interaction (P less than 0.025). Overall, opsonins were higher (P less than 0.05) in flushings of mares susceptible to endometritis than in flushings of mares resistant to endometritis, but this difference was only apparent at estrus. Preliminary characterization of opsonins in uterine secretions by ammonium sulfate fractionation and gel filtration indicated that opsonins were mainly associated with an ammonium sulfate-soluble fraction of high molecular weight (greater than 4 X 10(6] and an ammonium sulfate-precipitable fraction that was associated with immunoglobulin G.     

Immune enhancing effects of Lactobacillus acidophilus on Newcastle disease vaccination in chickens

Introduction and purposeDespite the various vaccination programs for protection against New Castle disease, it remains an important threat to the poultry industry. The ability of the probiotic bacteria to improve the immune system in both animals and humans supports their use as immune adjuvants for vaccination. The aim of the present study was to examine the effects of Lactobacillus acidophilus in ND vaccination.Materials and methodsA total of 170 one day old chicks were divided in 5 groups. In groups A, B and C chicks were received L. acidophilus (5 × 10⁹, 3 × 10⁹ and 2 × 10⁹) and also vaccinated with inactivated and attenuated ND vaccines. In group D, chicks only vaccinated without bacterial inoculation and group E was negative control with neither vaccine nor bacteria. Then IgG and HI NDV antibody titers were measured in all tested groups.ResultsIgG and HI NDV antibody levels were significantly higher in Lactobacillus treated groups especially in group A with 5 × 10⁹ bacteria, than only vaccinated and negative control groups. Also antibody levels against NDV increased during the vaccination period especially in probiotic treated groups.ConclusionIn conclusion, L. acidophilus can use for improving immunogenicity of NDV vaccination programs.     

The experimental infection of ponies with contagious equine metritis

Four pony mares were readily infected with the organism of contagious equine metritis by intracervical inoculation and one by coitus with an infected stallion. Infected mares developed an acute endometritis with local destruction of the endometrial epithelium. In 2 experimentally infected mares, infection appeared to have been spontaneously eliminated from the genital tract within 3 to 4 weeks. A third mare however remained persistently infected in the clitoral fossa over a long period and was a symptomless carrier. Four pony stallions were readily infected in the urethral fossa and the organism survived for varying periods without giving rise to any signs of infection. From 2 of these animals it appeared eventually to have been eliminated spontaneously. An experimentally infected stallion transmitted infection to a healthy mare by coitus. Bacteriological examination of infected pony stallions may occassionally give false negative results and fail to reveal the organism in the external genitalia. Repeated bacteriological examinations need to be undertaken before it can be concluded that a stallion is free of infection.     

鸭疫里默氏菌6型、12型与16型之间的交叉反应

鸭疫里默氏菌6型和12型仅在玻片凝集试验中存在较弱的交叉反应，这一交叉反应可经血清吸收试验得到消除；12型和16型在玻片凝集试验和试管凝集试验中均表现较强的双向交叉反应，但在琼扩试验中表现为较弱的单向交叉反应；6型和16型之间没有交叉反应。12型和16型之间的交叉反应和交叉沉淀反应均可经血清吸收后得以消除，但经过16型参考菌株吸收过的12型抗血清，还与6型菌株存在交叉凝集反应，只有同时用6型和16型菌株吸收，12型抗血清才具有血清型特异性。血清吸收对6型和12型抗血清的同型凝集反应能力和沉淀反应能力没有影响，但对16型抗血清的同源凝集能力和沉淀反应能力均有较大的影响，经、2型参考菌株吸收后，16型抗血清与同型抗原之间的凝集效价下降2个滴度，而且不再形成清晰的沉淀线。结果表明，6型和12型之间、12型和16型之间均存在ab-bc的抗原关系。     

Experimental Maedi Infection in Sheep: 2. Antibody Response

All 4 sheep inoculated via the respiratory tract with 7×10⁶ TCID50 af maedi M88 strain developed complement fixing (CF) antibodies within 3 months after inoculation, and a gradual rise in CF titers was found during the first year. The antibody titers have been maintained, though with some fluctation, through the following year, and the titers vary from 64 to 256. Virus neutralizing activity against maedi M88 strain was detected in the sera of all intrapulmonarily inoculated sheep within 8 months after inoculation. Titers have been maintained or have slightly increased. The level of titers, ranging from 8 to 256, was clearly different between individual sheep.One of the 4 sheep inoculated intracerebrally with 5×10⁵ TCID50 of maedi M88 strain developed CF antibodies 1 month after inoculation, but no neutralizing antibodies until death 11 months after inoculation. The rest of the intracerebrally inoculated sheep displayed no evidence of CF or neutralizing antibodies within 18 months after inoculation in spite of numerous virus isolations from peripheral blood leukocytes. The absence of antibodies might perhaps be attributed to phenomena such as differences in tropism, provirus state, immunological tolerance and size of inoculum.One sheep hyperimmunized with repeated s.c. and i.v. injections of maedi M88 strain developed high CF antibody titers but lower neutralizing antibody titers.The 2 uninoculated control sheep developed no CF or neutralizing antibodies within 18 months after inoculation.     

Homotypic and heterotypic serum and milk antibody to rotavirus in normal, infected and vaccinated horses.

The homotypic and heterotypic antibody response to rotavirus was determined in three pony mares and their foals. The normal concentrations of anti-rotavirus antibodies in mares' milk and mares' and foals' serum over the first 10 weeks post-partum were measured using IgA, IgG and rotavirus serotype-specific enzyme linked immunosorbent assays. Experimental infection of the foals with serotype 3 equine rotavirus produced a rapid, serotype-specific response which peaked 10 days after infection and a slower heterotypic response which peaked 32 days later. In contrast, vaccination of the mares with an inactivated, adjuvanted serotype 6 bovine rotavirus produced a heterotypic response similar to that of the homotypic response in both serum and milk, although the predominant response in serum was IgG, while in milk it was IgA. These results suggest that non serotype-restricted passive protection of foals against rotavirus may be achieved by parenteral vaccination of mares.     

Disinfection of the causative agent of contagious equine metritis by Nolvasan and Roccal II

Haemophilus equigenitalis, the proposed name for the bacterium that causes contaginous equine metritis (CEM) was tested for sensitivity to the disinfectant solutions Nolvasan® (2% chlorhexidine diacetate) and Roccal II® (10 alkyldimethylbenzylammonium chloride).Bacteria (10⁶) suspended in medium were inactivated in 10–20 min at 0°–2°C and in 2.5 min at 20°–22°C by a 3 : 128 dilution of Nolvasan. Roccal II, diluted 1 : 128, inactivated 10⁶ bacteria in 2.5 min at 0°–2°C and 20°–22°C.These dilutions of the disinfectants inactivated H. equigenitalis (titers of 0.1–11.0 × 10⁷/ml) in vaginal exudates from infected mares and pure cultures suspended in medium (titers of 1–5 × 10⁹/ml) in 10 min at 0°–2°C and 20°–22°C when the exudates and cultures were dried on metal carriers. The limit of detection of survivors was 3–32 bacteria/ml.It is recommended that contaminated instruments and other metal surfaces encountered during CEM infections should be decontaminated with either disinfectant for 10 min before rinsing.     

A study of Klebsiella pneumoniae infection in the uterus of the mare.

Two experiments incorporating 13 mares were conducted for the purpose of producing and monitoring intrauterine infection with Klebsiella pneumoniae. In the pilot study, the infection was produced with strains of K pneumoniae type 68 and type 10 isolated from the genital tract of stallions with a history of breeding problems. In the principal study, K pneumoniae type 68 was used to produce the infection. Tampons and guarded culture swabs were used to obtain uterine samples in the pilot study. In comparing the efficacies of isolation of K pneumoniae with the tampons and isolation with standard guarded culture swab, the tampon proved to be a more reliable means with which to isolate K pneumoniae and was used in the principal study. In both studies, inoculated mares became infected and remained infected at least until the postinoculation estrous cycle was initiated or was completed. Some of the inoculated mares remained infected through more than one estrous cycle. The numbers of K pneumoniae decreased in the uterus of mares after completing the estrous cycle after inoculation. Klebsiella pneumoniae was not demonstrable in frozen tissue sections of uterine biopsy specimens stained by fluorescent antibody technique. Postinoculation sera antibody titers to K pneumoniae, as determined, using the capsule swelling technique, were no higher than 1:8.     

The Antibody Response in Pigs Inoculated with Attenuated African Swine Fever Virus

Pigs were inoculated with a modified isolate of African swine fever virus (ASFV). Complement-fixing (CF) and agar gel diffusion precipitin (AGDP) antibodies could be detected in the serums of most pigs from 14-days postinoculation (DPI) until their immunity was challenged with virulent ASFV at 117 DPI.

Reductive cleavage with 2-mercaptoethanol showed that serums collected at 14 to 35 DPI contained 19S antibody, but that the 7S antibody was dominant at 35 and 117 DPI. This distribution of antibody was confirmed by sucrose-gradient centrifugation. Nearly all of the early serums also contained 7S antibodies which fixed complement and reacted in the AGDP test. Pigs whose serums contained both CF and AGDP antibodies at time of challenge failed to develop acute disease while pigs without CF antibodies were usually not protected.

Pigs surviving challenge with virulent virus showed no increase in antibody titers, or reversion to 19S antibody.

     

Experimental caprine coccidiosis caused by Eimeria arloingi: Morphopathologic and electron microscopic studies

The progressive morphohistopathologic changes, distribution pattern of lesions and ultrastructural characteristics in Eimeria arloingi infection were investigated in experimentally infected kids. The 18 newborn animals allocated to 3 equal groups. Two of groups, A, B were inoculated with a single dose of 1 × 10³ and1 × 10⁵ sporulated oocysts of E. arloingi, respectively. At 7, 14, 21, 28, 35, and 42 days postinoculation (DPI), 1 kid from each group was necropsied for pathologic and ultrastructural studies. Progressive lesions were present at 21, 28, 35 and 42 DPI in the jejunum, ileum, cecum with fewer in the duodenum and proximal colon. The oocysts shedding begin between 16 to 18 DPI. Grossly, minimal changes were observed at 21 DPI as few whitish plaques or nodules and advanced lesions at 42 DPI as pseudoadenomatous pattern in the mucosa and a cerebriform pattern on the serosal surface of jejunum and ileum. Early histopathologic lesions due to schizogony phase were including presence of intracytoplasmic developmental stages of the parasite such as trophozoites, immature to mature schizonts and mild infiltration of inflammatory cells. In late lesions due to various stages of gametogony, the histological pattern was mainly remarkable hyperplasia of the villi and crypts epithelial cells, eventually developed into papillary projections of reactive epithelium. The mesenteric lymph nodes showed a few numbers of large schizonts in the cortical lacteals. This study showed E. arloingi as a highly pathogenic species for kids, the incubation period was 16–18 days and the main target organ was jejunum with characteristic morphohistopathologic lesions.     

Passive transfer of Theileria equi antibodies to neonate foals of immune tolerant mares

Equine babesiosis, a tick transmitted haemoprotozoan disease caused by Theileria equi is globally distributed and responsible for heavy economic losses to the equine husbandry. Equids reared in endemic areas usually pick up infection at an early age and become immune tolerant throughout their life span. We studied the level of passively transferred antibodies in neonate foals born from pre-immuned mares. Latently T. equi infected pre-immuned pony and donkey mares (three each) were selected and T. equi antibody titres in neonates was monitored till 90 days post foaling (DPF) by applying Dot-ELISA on sequentially collected serum samples from foals and their dams. A very high antibody titre was observed in pre-immuned pony and donkey mares. The maximum antibody of 1:60 to 1:80 was observed in pony's and donkey's foal on 2–16 and 2–10 DPF, respectively and thereafter it declined to less than 1:20 on 63–77 and 56–63 DPF. Simultaneously parasite carrying status in neonate foals and their dam was also monitored by applying PCR on blood samples. We could demonstrate PCR amplification in dam's blood samples while no amplification was recorded in neonate's blood samples. This study indicated that new-born foals were born naïve and passively transferred immunity was transitory which wanes after 63–77 DPF.     

Comparisons of the complement-fixation, indirect fluorescent antibody, and card agglutination tests for the diagnosis of bovine anaplasmosis.

Results of complement-fixation (CF), indirect fluorescent antibody (IFA), and card agglutination (CT) tests were statistically compared, using 380 serum samples obtained from 140 cattle which were disease-free or naturally or experimentally infected with Anaplasma marginale of Colombian origin. The IFA test was significantly the most sensitive for detection of amimals infected with anaplasmosis (97%); the CT test and the CF test were less so (84% and 79%, respectively). However, the most efficient test for identifying noninfected animals was the CF test (100%), and the CT and the IFA tests were less efficient (98% and 90%). A linear regression analysis performed on the average IFA and CF titers of 10 calves artificially infected with A marginale during a 20-week period showed significant regression coefficients for both tests. The regression line for the CF titers decreased below the sensitivity threshold at 14 weeks after calves were inoculated, whereas the regression line for the IFA titers continued above the sensitivity threshold 20 weeks after inoculation. The CT test also detected antibodies until the end of the observation period.     

Leptospira interrogans serovar pomona vaccines with different adjuvants in cattle

Three groups of four heifers were vaccinated twice, 11 weeks apart. One group received a commercial pomona vaccine with aluminium hydroxide adjuvant, the second a similar experimental vaccine, and the third a Freund's compete adjuvant (FCA) vaccine. After 47 weeks, the heifers were challenged with at least 65 × 10⁸ virulent serovar pomona organisms.

All vaccinated animals resisted the challenge, and leptospirae were only found in urine from unvaccinated controls.

The outcome of the challenge was not predictable from microscopic agglutination, cold and warm complement fixation, and growth inhibition titres.

The FCA vaccine gave rise to considerably higher antibody responses than the two aluminium hydroxide vaccines, which gave similar responses.     

Measurement of total volume and protein concentration of intrauterine secretion after intrauterine inoculation of bacteria in mares that were either resistant or susceptible to chronic uterine infection.

Undiluted uterine secretion was used to determine the concentration of total protein and the accumulated volume of uterine secretion after a bacterial inoculation in mares susceptible and resistant to chronic uterine infection (CUI). The uterus of 6 susceptible and 5 resistant mares was inoculated with 5 x 10(6) Streptococcus zooepidemicus on the third day of estrus. Using a tampon inserted in the uterus, secretions were sampled at 5, 12, 24, and 36 hours after inoculation, followed by intrauterine lavage with phosphate buffered saline solution. The concentration of protein was determined in the undiluted secretion as well as in the uterine washing and the total amount of accumulated uterine secretion was calculated. Protein concentrations in plasma were compared before and after absorption by the tampon. Protein concentration of plasma before and after absorption by the tampon did not differ. Mares susceptible to CUI accumulated significantly (P less than 0.001) more fluid in the uterus than mares resistant to CUI, and uterine washings from the resistant mares were significantly (P less than 0.05) more dilute than those from the susceptible mares. Significant differences in protein concentrations between susceptible and resistant mares were not found. It was concluded from this study that the described method to sample undiluted uterine secretion was practical and reliable for the analysis of protein concentration. Various concentrations of uterine secretions in washings from susceptible and resistant mares emphasizes the importance in using undiluted uterine secretions or dilution markers in washings when intrauterine products are analyzed.     

Feed preferences of ponies

Preference trials were conducted with mature ponies. In Trial 1, oats were compared with oats plus sucrose. Four of six pony geldings selected oats plus sucrose, but one pony demonstrated a dislike for sucrose and one selected from the bucket on the right side regardless of content. Oats, maize, barley, rye and wheat were compared in Trial 2 using six mature pony mares. Oats were the preferred grain, with maize and barley ranking second and third respectively. Wheat and rye were the least preferred. Even though the ponies demonstrated preference, the total intake at a given meal was not greatly depressed when only the less palatable grains were fed. In Trial 3, pony mares selected a diet containing 20 per cent dried distillers' grain and 80 per cent of a basal mixed diet of maize, oats, wheat bran, soybean meal, limestone and molasses over 100 per cent basal mixed diet, but selected the basal diet over diets containing 20 per cent blood meal, beet pulp or meat and bone meal and 80 per cent basal diet. They did not differentiate against diets containing 20 per cent alfalfa meal or 10 or 5 per cent meat and bone meal when the diets were compared to the basal mixed diet.     

Comparison of the pathogensis of two isolates of <Emphasis Type="Italic">Besnoitia caprae</Emphasis> in inbred BALB/c mice

This study was performed to evaluate the infectivity of bradyzoites of two Besnoitia caprae isolates, BC-1 and BC-2, to inbred BALB/c mice. Each group of inbred BALB/c mice was inoculated intraperitoneally with 1 × 10³, 1 × 10⁴, 1 × 10⁵, 5 × 10⁵ and 1 × 10⁶ of one of the two isolates of B. caprae bradyzoites. The mice were monitored daily for a period of 40 days for survival. After death of each mice, several passages from its peritoneal washing and tissues were analyzed using ribosomal DNA-specific PCR assay. Marked differences in pathogenicity between the isolates were seen. All the inbred BALB/c mice infected with BC-2 survived but all the mice that were administered with 1 × 0⁵, 5 × 10⁵ and 1 × 10⁶ BC-1 bradyzoites were died within 4–9 days post-infection (DPI). Histopathological examination of the tissues of the dead mice revealed hyperemia and necrosis with presence of mononuclear and polymorphonuclear cell infiltration in myocardium, spleen and intestines together with interstitial pneumonia and peritonitis. All inbred BALB/c mice in the 1 × 10³ and 1 × 10⁴ groups of BC-1 inoculated mice survived and they were euthanized after 40 DPI. Chronic inflammation with infiltration of mononuclear cells was evident in myocardium, spleen, alveolar septa of the lungs of most of the examined tissues with hemorrhagic enteritis in the mice infected with 1x10⁶ bradyzoites. The mice infected with different doses of BC-2 were euthanized after 40 DPI and no lesion was seen in histopathological sections of their organs. All peritoneal washings and examined tissues were PCR positive in BC-1 group. This experiment is the first report to show inbred BALB/c mice as a relevant model for B. caprae and demonstrates that this strain of inbred BALB/c mice is a suitable animal model for biological studies and examination of pathogenesis for this species of Besnoitia. The present findings also provide evidence for significant differences between the two isolates of B. caprae.     

Endometritis in the mare caused by a Coryneform organism--a case report and experimental studies

Persistent purulent endometritis in a mare was attributed to an unclassified species of Corynebacterium. Following intrauterine infusions of 20% betadine for 5 days the purulent vulval discharge ceased and the mare appeared clinically normal. Based on histological examination of endometrial biopsy samples, the severe acute inflammatory reaction had largely resolved 2 days after therapy. Three maiden mares considered resistant to bacterial endometritis received single intrauterine inoculations of 1.8 X 10(9) colony-forming units of the Corynebacterium species. The uterine response was followed by vaginal speculum examinations, uterine cultures and cytology, and endometrial histology. After an acute inflammatory reaction, each mare had recovered completely within 2 weeks. Most rapid recovery occurred in the mare in estrus at the time of inoculation. Subsequent secondary infections were detected in two mares. The uncertainty of correlations between results obtained by various diagnostic techniques emphasized the problems associated with each. This report illustrates the concept that endometritis in individual mares may relate more to as yet unidentified "mare factors" controlling uterine defense than to primary invasion by bacteria.