Bacterial Agglutination Titre Research Articles

Immuno-pathological changes in Indian catfish Clarias magur (Hamilton, 1822) upon experimental challenge with Aeromonas hydropila

Motile Aeromonas septicaemia (MAS), caused by Aeromonas hydrophila, is one of the most significant problem among the bacterial diseases responsible for causing mortality in catfishes. The objective this study was to understand the modulation of innate immune parameters and histopathological alteration in Indian catfish Clarias magur (Hamilton, 1822), following intraperitoneal injection with A. hydrophila. At 3 to 8 h post-infection (hpi), respiratory burst activity, myeloperoxidase, protease, total anti-protease and α2 macroglobulin increased significantly (p<0.05) indicating immune response of host against pathogen invasion at an early stage of infection. The total protein, albumin, albumin/globulin ratio and bactericidal activity were significantly decreased indicating the response of pathogen to suppress host immune response to establish infection. Serum bactericidal activity, bacterial agglutination titre and lysozyme activity increased significantly at 24 hpi. Histopathological examination of infected fish revealed the accumulation of melanomacrophage centres, congestion, hepatocyte degeneration and vacuolated hepatocytes of liver; hyaline droplet accumulation, lymphocytes infiltration and diffuse necrosis of renal tubule in kidney; unilateral fusion of secondary gill lamellae, leukocytic infiltration as well as dilated central venous sinus in gills. Knowledge regarding the immune response in magur could be useful for developing strategies for improving disease resistance against A. hydrophila infection. Keywords: Aeromonas hydrophila, Histopathological alterations, Indian catfish, Innate immune parameters, Intraperitoneal challenge

Parenteral immunization of PLA/PLGA nanoparticle encapsulating outer membrane protein (Omp) from Aeromonas hydrophila: Evaluation of immunostimulatory action in Labeo rohita (rohu)

Advanced vaccine research approaches needs to explore on biodegradable nanoparticles (NPs) based vaccine carrier that can serve as antigen delivery systems as well as immuno-stimulatory action to induce both innate and adaptive immune response in fish. Immunogenicity of PLA and PLGA NPs encapsulating outer membrane protein (Omp) antigen of Aeromonas hydrophila were evaluated through intra-peritoneal injection in fish, Labeo rohita. Antigen loaded PLA-Omp (223.5 ± 13.19 nm) and PLGA-Omp (166.4 ± 21.23 nm) NPs were prepared using double emulsion method by efficiently encapsulating the antigen reaching the encapsulation efficiency 44 ± 4.58% and 59.33 ± 5.13% respectively. Our formulated PLA Omp and PLGA-Omp NPs were in nanometer range (<500 nm) and could be successfully endocyted in the body. Despite low antigen loading in PLA-Omp, it showed considerably slower antigen release in vitro than PLGA-Omp NPs. Other physical properties like zetapotential values and poly dispersity index (PDI) confirmed the stability as well as monodisperse nature of the formulated nanoparticles. The spherical and isolated nature of PLA-Omp and PLGA-Omp NPs were revealed by SEM analysis. Upon immunization of all antigenic formulations (PLA-Omp NP, PLGA-Omp NP, FIA-Omp, PLA NP, PLGA NP, PBS as control), significant higher bacterial agglutination titre and haemolytic activity were observed in case of PLA-Omp and PLGA-Omp immunized groups than rest groups at both 21 days and 42 days. The specific antibody response was significantly increased and persisted up to 42 days of post immunization by PLA-Omp, PLGA-Omp, FIA-Omp. PLA-Omp NPs showed better immune response (higher bacterial agglutination titre, haemolytic activity, specific antibody titre, higher percent survival upon A. hydrophila challenge) than PLGA-Omp in L. rohita confirming its better efficacy. Comparable antibody response of PLA-Omp and PLGA-Omp with FIA-Omp treated groups suggested that PLA and PLGA could be replacement for Freund's adjuvant (for stimulating antibody response) to overcome many side effects offering long lasting immunity. Our encouraging results suggest that PLA/PLGA nanoparticles based delivery system could be a novel antigen carrier for parenteral immunization in fish.

Poly D, L-lactide-co-glycolic Acid (PLGA)-encapsulated CpG-oligonucleotide (ODN) on Immune Response in Cyprinus carpio against Aeromonas hydrophila

The effect of Poly D, L-lactide-co-glycolic acid (PLGA) encapsulated ODN 1668 nanospheres (NS) on innate and adaptive immune response in common carp, Cyprinus carpio against Aeromonas hydrophila is reported. PLGAencapsulated ODN biodegradable NS did not have a significant impact on particle size as expected of the particles was clearly positive for non-coated and coated PLGA/ODN NS. The myeloperoxidase (MPO) activity and serum haemolysin titre significantly increased in fish injected with ODN 1668 and PLGA-encapsulated ODN 1668 groups on week 4 whereas the respiratory burst (RB) activity significantly increased when administered with ODN 1668 and PLGA-encapsulated ODN 1668 (PLGA-ODN 1668) from weeks 1 to 4. The haemoagglutinating titre was significantly enhanced in PLGA-encapsulated ODN 1668 group on week 2 while in ODN 1668 and PLGA-encapsulated ODN 1668 groups the increase manifested on week 4. The bacterial agglutination titre significantly increased in ODN 1668 and PLGA-encapsulated ODN 1668 groups on weeks 2 and 4. The cumulative mortality in ODN 1668 and PLGAencapsulated ODN 1668 groups were 10% each whereas it was 15% in PLGA group. This study indicates that single administration of PLGA-encapsulated ODN 1668 nanospheres elicits better immune response than PLGA or ODN 1668 alone in C. carpio against A. hydrophila.

Immunostimulatory Activities of a Decapeptide Derived from Alcaligenes faecalis FY-3 to Crucian Carp

A strain was isolated from a soil sample collected from Weihe river in Shaanxi province (108°03'E 34°14'N), which was identified as Alcaligenes faecalis by 16S rRNA analysis. A compound M showing potent immune activity was isolated from secondary metabolites of the strain through bioassay-guided isolation techniques. The structure of the compound M was elucidated using FT-IR, EI-MS, 1H NMR and 13C NMR spectra and identified as cyclo-(L-Pro-Gly)5 which was first time reported as a natural product. We evaluated the immune effects of the cyclo-(L-Pro-Gly)5 on the basis of serum lysozyme activity, bacterial agglutination titre assay, superoxide anion production and phagocytic activity assay, and they were found to be significantly increased by cyclo-(L-Pro-Gly)5. The effects of cyclo-(L-Pro-Gly)5 on immune-related gene expression were further investigated. The outcomes of real-time quantitative polymerase chain reaction (RQ-PCR) proved that the transcribing level of interleukin 6β (IL-6β) and inducible nitric oxide synthase 1β (iNOS-1β) mRNA in the blood leucocytes have been augmented by cyclo-(L-Pro-Gly)5. The challenge experiment showed that crucian carp injected the cyclo-(L-Pro-Gly)5 had significantly (P < 0.05) lower cumulative mortality (13.0%) compared with the control (45.4%) after infection with live Aeromonas hydrophila. These results suggested that cyclo-(L-Pro-Gly)5 is a possible immunostimulant and may strengthen the immune response and protect the heath status of crucian carp against A. hydrophila.

Family association between immune parameters and resistance to Aeromonas hydrophila infection in the Indian major carp, Labeo rohita

Seven innate immune parameters were investigated in 64 full-sib families (the offspring of 64 sires and 45 dams) from two year-classes of farmed rohu carp ( Labeo rohita). Survival rates were also available from Aeromonas hydrophila infection (aeromoniasis) recorded in controlled challenge tests on a different sample of individuals from the same families. Due to strong confounding between the animal additive genetic effect and the family effects (common environmental + non-additive genetic), reliable additive (co)variance components and hence heritabilities and genetic correlations could not be obtained for the investigated parameters. Therefore, estimates of the association of challenge test survival with the studied immune parameters were obtained as product moment correlations between family least square means. These correlations revealed statistically significant ( p < 0.05) negative correlations of survival with bacterial agglutination titre (−0.48), serum haemolysin titre (−0.29) and haemagglutination titre (−0.34); and significant positive correlation with ceruloplasmin level (0.51). The correlations of survival to aeromoniasis with myeloperoxidase activity, superoxide production and lysozyme activity were found to be not significantly different from zero ( p > 0.05). Assuming that the negatively correlated candidate traits are not favourable as indirect selection criteria, the results suggest that ceruloplasmin level could potentially be a marker for resistance to aeromoniasis in rohu. The use of this immune parameter as an indirect selection criterion for increased resistance to aeromoniasis in rohu will, however, require that the parameter shows significant additive genetic variation and a significant genetic correlation with survival. Further studies are therefore needed to obtain a reliable heritability estimate for ceruloplasmin and its genetic correlation with survival from aeromoniasis.

Application of PCR-RF-SSCP to study major histocompatibility class II B polymorphism in common carp ( Cyprinus carpio L.)

A variety of methods have been applied for the characterization of major histocompatibility (MH) polymorphism in fish. We optimized a technique designated polymerase chain reaction-restriction fragments-single strand conformation polymorphism (PCR-RF-SSCP) for screening a large number of individuals for the Cyca-DAB1 and Cyca-DAB2 genes polymorphism in common carp. The advantages of this technique are simplicity, high sensitivity and low costs. PCR-RF-SSCP analysis revealed different genotypes consisting of unique combinations of the Cyca-DAB1 and Cyca-DAB2 sequences with the number of SSCP bands clearly correlating with the degree of heterozygosity of the Cyca-DAB1 and Cyca-DAB2 genes. We found four alleles for Cyca-DAB1 ( *02-*05) gene but only one allele for Cyca-DAB2 ( *02) and noted that the Cyca-DAB2 gene was either homozygous or absent. PCR-RF-SSCP analysis of n = 79 carp individuals challenged with Aeromonas hydrophila indicated that individuals bearing no Cyca-DAB2 gene showed higher cumulative mortality and lower bacterial agglutination titers during the experiment. We suggest that our PCR-RF-SSCP method can be used to study correlations of different MH class II B genotypes/alleles with resistance of common carp to specific pathogens.

Dietary levamisole modulates the immune response and disease resistance of Asian catfish Clarias batrachus (Linnaeus)

In order to determine the immunomodulatory effect of dietary levamisole in Asian catfish (Clarias batrachus), fish were fed four different diets for 10 days: a formulated diet as control and the same diet supplemented with 50, 150 or 450 mg levamisole kg−1 feed. The serum bacterial agglutination titre against Aeromonas hydrophila as a measure of specific immunity, serum haemagglutination titre, natural haemolytic complement activity (ACH50), myeloperoxidase and lysozyme activities, total protein level and oxidative radical production by neutrophils as a measure of non-specific immunity as well as disease resistance against A. hydrophila challenge to separate vaccinated and non-vaccinated groups were evaluated at 0, 1, 2 and 3 weeks after last administration of levamisole. Levamisole supplement at the lowest level (50 mg kg−1) significantly enhanced oxidative radical production and serum myeloperoxidase (MPO) content immediately after 10 days of feeding, which reached peak values after 3 and 2 weeks of feeding respectively. Haemolytic complement and haemagglutination titre were significantly enhanced after 3 and 1 weeks respectively. Haemolytic complement activity and MPO activities were significantly raised to 150 mg kg−1 after 3 and 2 weeks, respectively. At the highest level of levamisole feeding (450 mg kg−1) significant decreases in superoxide production and complement activity were measured immediately after levamisole feeding, which returned to the normal level after 1 week post- feeding. Fish were challenged with a virulent strain of A. hydrophila at 0, 1, 2 and 3 weeks after levamisole feeding, and the cumulative per cent survival was recorded over 10 days. Feeding levamisole at 50, 150 or 450 mg kg−1 increased per cent survival in vaccinated fish immediately after levamisole feeding, and survival was significantly higher at 450 mg kg−1. There was no difference in mortality patterns in non-vaccinated fish. The results support the use of levamisole at 50 mg kg−1 feed for 10 days as an immunostimulant in Asian catfish farming.

Dietary immunostimulants influence specific immune response and resistance of healthy and immunocompromised Asian catfish Clarias batrachus to Aeromonas hydrophila infection

In order to determine the efficacy and immunoreversal effect of the 4 dietary immunomodulators, viz. lactoferrin, beta-1,3 glucan, levamisole and vitamin C, on disease resistance of a commercially important catfish, Clarias batrachus, fish were fed diets supplemented with various levels of these substances in 2 subgroups, healthy and immunocompromised, during a 30 d trial. An artificial immunosuppressive state was induced by giving 3 intraperitoneal (i.p.) injections of cyclophosphamide (CYP) at a dose level of 200 mg kg(-1) body weight at 1 wk intervals in the immunocompromised vaccinated subgroup and 3 consecutive injections 3 d before challenge in the immunocompromised non-vaccinated subgroup. On the first day of the experiment, the fish were vaccinated against a formalin-killed Aeromonas hydrophila bacterin. After 30 d, antibody titre (as measured through bacterial agglutination titre) and disease resistance against A. hydrophila were determined. The results demonstrate that all 4 immunomodulators were capable of significantly (p < 0.05) enhancing the specific immune response; this was evident through raised antibody titre and protection against A. hydrophila in both healthy and immunocompromised vaccinated subgroups compared to their respective controls. Similarly, all 4 substances significantly raised the survival rates in immunocompromised and healthy non-vaccinated fish. Thus, these substances were capable of reducing the immunosuppression induced by CYP injections in both vaccinated and non-vaccinated fish compared to their respective controls. Among the 4 substances studied, beta-1,3 glucan was found to be the most effective immunomodulator, followed by levamisole, lactoferrin and vitamin C in Asian catfish. Therefore, the results support the introduction of these substances into the diet of fish grown in farms under immunosuppressive/stressful conditions in order to enhance protection against infection and offer economic benefits.

Effects of cyclophosphamide on the immune system and disease resistance of Asian catfish Clarias batrachus

Cyclophosphamide (CYP), a multifunctional alkylating agent is known as a potent immunosuppressor in endotherms. Here, an experiment was conducted in an ectothermic Asian catfish species, Clarias batrachus to investigate its effect on non-specific and specific immunity as well as disease resistance against a common bacterial pathogen challenge. CYP was intraperitoneally injected for 3 consecutive days at a rate of 200 mg kg −1 body weight. After 72 h post-injection, control and CYP-treated fish were screened for superoxide production through nitroblue tetrazolium (NBT) assay, myeloperoxidase (MPO) activity, packed cell volume (PCV) and total protein, lysozyme, alternative complement activity (ACH50) and natural haemagglutinin titre as a measure of non-specific immunity level as well as disease resistance against Aeromonas hydrophila challenge to vaccinated and unvaccinated fish. To study the effect on specific immunity, CYP was injected thrice at an interval of 7 days after bacterin injection and serum antibody titre was measured by bacterial agglutination titre assay. The results showed a significant ( P < 0.05) decrease in NBT and MPO activities, and percent survival against A. hydrophila challenge (both in unvaccinated and vaccinated fish) in CYP-treated fish, when compared to control fish. The above results support the immunosuppressive action of CYP in freshwater catfish, C. batrachus. The neutrophil activities, as measured through superoxide production and myeloperoxidase levels, might be important contributors during A. hydrophila infection and that treatment with CYP reduces phagocytic killing power and inhibits resistance against aeromoniasis.

Dietary bovine lactoferrin induces changes in immunity level and disease resistance in Asian catfish Clarias batrachus

The effects of including bovine lactoferrin (Lf) in the diet of the Asian catfish (Clarias batrachus) on specific and non-specific immunity as well as disease resistance were investigated. The catfish were fed four different diets for 2 weeks: a commercial diet as control and the same diet supplemented with 50, 100 and 200mgbovineLf/kg feed. After 1 and 2 weeks, serum bacterial agglutination titre against Aeromonas hydrophila as a measure of specific immunity; natural serum haemolysin titre, lysozyme activity and oxidative radical production by neutrophils as a measure of non-specific immunity as well as disease resistance against A. hydrophila challenge to vaccinated and non-vaccinated animals were evaluated. The results showed that Lf supplements, particularly at 100mg level, significantly (P<0.05) enhanced serum lysozyme level, oxidative radical production and level of protection against A. hydrophila challenge in non-vaccinated animals irrespective of length of exposure. The specific immunity was not influenced by Lf feeding as evidenced from the bacterial agglutination titre and level of protection in vaccinated animals. As Lf feeding at 100mg/kg for 1 week is able to enhance the non-specific immunity and disease resistance of catfish efficiently, these results support the possible use of Lf as an immunostimulant for farmed catfish.

Influence of high dietary α-tocopherol intakes on specific immune response, nonspecific resistance factors and disease resistance of healthy and aflatoxin B1 -induced immunocompromised Indian major carp, Labeo rohita (Hamilton)

The effect of aflatoxin treatment and/or feeding of a high level of alpha-tocopherol on immune response and disease resistance was investigated in Indian major carp, Labeo rohita. Group A served as a healthy control, group B was treated with aflatoxin, group C was fed a high level of alpha-tocopherol whereas group D was exposed both to aflatoxin and a high level of dietary alpha-tocopherol for 60 days. Aflatoxin B1 (AFB1) was injected once intraperitoneally into fish on the first day of the experiment (groups B & D). High levels of DL-alpha-tocopherol (1000 mg kg(-1) feed) were provided to healthy as well as AFB1-treated immunocompromised fish for 60 days (groups C & D). At the end of the experiment blood samples were assayed for changes in nonspecific immunity and humoral protein levels. Disease resistance against two common bacterial pathogens viz., Aeromonas hydrophila and Edwardsiella tarda were evaluated in all groups. Significant (P < 0.05) suppression of specific immunity as measured through haemagglutination (HA) titre against sheep red blood cells (SRBCs) as well as bacterial (formalin-killed E. tarda) agglutination titre; nonspecific resistance factors viz., globulin level, serum bactericidal and lysozyme activities, neutrophil activities, and disease resistance against two bacterial pathogens only in aflatoxin-treated fish with respect to the control group, clearly indicated the immunosuppressive nature of aflatoxin. Feeding of a high level of alpha-tocopherol to AFB1-treated immunocompromised fish significantly (P < 0.05) raised specific immunity, nonspecific resistance factors and disease resistance capacity when compared with aflatoxin-exposed fish. Disease resistance and enhancement of immune status through feeding of high levels of alpha-tocopherol to healthy as well as AFB1-treated immunocompromised fish confirmed the potential of alpha-tocopherol in carp feed for prevention of disease and for combating natural/environmental immunosuppressants.

The effect of dietary immunomodulation upon Edwardsiella tarda vaccination in healthy and immunocompromised Indian major carp (Labeo rohita)

In order to determine the impact on disease resistance of four dietary immunomodulators viz., β-1,3 glucan, levamisole, vitamins C and E, in an important farmed Indian major carp species, rohu (Labeo rohita Ham.), fish were fed diets containing various levels of these substances during a 60 day trial. Aflatoxin B1(AFB1) at 1·25mg kg−1body weight was injected intraperitoneally (i.p.) into fish to induce an immunosuppressive state on the first day of the experiment in some individuals. The fish were vaccinated against formalin-killedEdwardsiella tarda vaccine on day 30 of the experiment. Specific immunity, as measured by bacterial agglutination titre and disease resistance against E. tarda, was determined at the end of the trial. The results demonstrate that all the four immunomodulators were capable of significantly (P< 0·05) increasing specific immunity and reducing mortality in immunocompromised fish but failed to enhance specific immunity and protection in healthy fish. The increased bacterial agglutination titre by β-1,3 glucan, and reduced mortality losses by both β-1,3 glucan and levamisole were marked in healthy vaccinated fish compared with their controls. Similarly, all four substances significantly reduced the mortality rates in immunocompromised and healthy unvaccinated fish. Out of these four substances, glucan was recorded to be the most effective immunomodulator in rohu. The present results suggest that the introduction of these substances into the diet of fish grown in farms under immunosuppressive/stressful conditions could increase their resistance to infection by reducing mortality rates and offer economic benefits.

Effect of dietary β-1,3 glucan on immune responses and disease resistance of healthy and aflatoxin B1-induced immunocompromised rohu (Labeo rohita Hamilton)

The immunostimulant β-1,3 glucan was fed at 0·1% in feed for 7 days to healthy and aflatoxin B1(AFB1)-induced immunocompromised fish, Labeo rohita (one of the major tropical carp species), in a 60 day trial. The effects of AFB1, glucan and their interactions on non-specific and specific immunity levels and disease resistance of fish were studied. A single intraperitoneal injection of AFB1at 1·25mg kg−1body weight) caused a significant (P< 0·05) reduction in non-specific immunity as measured through neutrophil phagocytic indices, serum bactericidal activity, and specific immunity as measured through bacterial agglutination titre against Edwardsiella tarda, as well as reduced protection against Aeromonas hydrophila challenge in comparison to control fish which were exposed neither to aflatoxin nor to glucan. Feeding of glucan to healthy fish raised the non-specific and specific immunity level and protection against bacterial infection compared with the control. Feeding of glucan to AFB1-induced immunocompromised fish for 7 days significantly raised the degree of resistance against A. hydrophila challenge and the non-specific immunity level in comparison to non-treated AFB1exposed fish. Although feeding of glucan was able to increase specific immunity, al measured through haemagglutination titre against sheep red blood cells, and bacterial (E. tarda) agglutination titre in healthy fish in comparison to all other groups, no significant increase in specific immunity to the aflatoxin-exposed group was seen.

Immunosuppressive effects of aflatoxin B 1 in Indian major carp ( Labeo rohita)

Aflatoxin B 1 (AFB 1) is a potent immunomodulator in endotherms. The experiment was carried out to study the immunosuppressive nature of AFB 1 in one ectothermic species of Indian major carp. Graded levels (0, 1.25, 5.00 mg/kg of body weight) of purified AFB 1 were intraperitoneally (i.p.) injected into rohu ( Labeo rohita) fingerlings weighing 30–50 g, and the fish were observed for a period of 90 days. At the end of the trial, blood samples were collected from the control group as well as the AFB 1 injected fish and were screened for nitroblue tetrazolium (NBT) assay, serum total protein, albumin, globulin, albumin–globulin ratio (A:G), serum bactericidal activity and bacterial agglutination titre against Edwardsiella tarda. The aflatoxin-treated fish revealed a reduction of total protein, globulin levels, bacterial agglutination titre, NBT and serum bactericidal activities, as well as an enhanced A:G ratio without change in albumin concentration, irrespective of dose levels of toxin treatment, when compared to the control group. Thus, AFB 1 proved to be immunosuppressive in rohu even at the lowest dose (1.25 mg/kg body weight) of toxin treatment. This could be of economic significance in intensive culture systems of rohu.

Sensitivity of a standard host resistance assay using Streptococcus agalactiae for assessing exposure to immunotoxicants in wild cotton rats (Sigmodon hispidus).

Resident small mammals have been used for in situ biomonitoring of contaminated waste sites containing suspected immunotoxicants. Host resistance assays, which involve challenging animals with an actual pathogen, allow for testing of overall immune system function in animals. Because such assays have not been evaluated for use with wild rodent species, it was our objective to assess the efficacy of Streptococcus agalactiae as a pathogenic model for use in a host resistance assay for detecting alterations in immune system function in wild cotton rats (Sigmodon hispidus). The ability of the assay to detect immunosuppression was evaluated by inducing immunosuppression chemically (cyclophosphamide or dexamethasone) and by protein malnutrition. The estimated lethal dose of S. agalactiae that killed 50% of challenged animals (LD50) was 5.76x10(7) colony-forming units (CFUs). Although bacterial agglutination titers indicated that animals developed an antibody response when immunized, immunization was not sufficient to adequately protect animals from a subsequent pathogenic challenge. Sensitivity of the host resistance assay was only suitable for detecting substantial immunosuppression, such as that induced by protein malnutrition or dexamethasone administration.

The immune response to experimental Pasteurella haemolytica infection in calves

Four male dairy calves, ages 1–9 months, were inoculated intratracheally (IT), with log dilutions (1.5×10 3−1.5×10 6) of an isolate of P. haemolytica A-1. Doses of bacteria varied according to ages of the calves, older calves receiving the larger doses. All four calves became severely ill within 24 h after inoculation and antibiotic treatment was considered essential. Two months later the four calves remained healthy after IT injection of P. haemolytica, again given in log dilution (2.8×10 2−2.8×10 5). The control calf, given a dilution of only 28 viable P. haemolytica (plate count), developed severe respiratory infection 9 days post inoculation. Antibiotic treatment was given to this calf for 7 days, at which time recovery was evident. All five calves developed direct bacterial agglutination titers to P. haemolytica. Persistent leukocyte migration inhibition indexes of all calves were decreased by ⩾20% compared to their controls. Although the initial doses administered were low, the calves became ill. Most reports refer to massive doses necessary to produce primary disease and significant agglutination titers.

Demonstration of antibodies against Yersinia enterocolitica lipopolysaccharide in human sera by enzyme-linked immunosorbent assay.

Antibodies against Yersinia enterocolitica serotype O:3 lipopolysaccharide present in sera from patients with Yersinia infection were studied by using an enzyme-linked immunosorbent assay. Of the sera with significant bacterial agglutination titers against Y. enterocolitica type O:3, 86% contained anti-lipopolysaccharide antibodies of the immunoglobulin G class. With the sera of some patients, we demonstrated increasing anti-lipopolysaccharide antibody levels of immunoglobulin G class in spite of decreasing bacterial agglutination titers. The assay was specific for lipopolysaccharide from Y. enterocolitica type O:3, and in inhibition experiments lipopolysaccharide could be detected in amounts of greater than or equal to 0.5 micrograms/ml.

An agglutination test for the serodiagnosis ofCorynebacterium sepedonicum

Antisera specific forCorynebacterium sepedonicum were produced in three rabbits and in a goat. Bacterial cell agglutination titers of 640, 1280, and 1280 were recorded for the rabbit sera and 320 for the goat antiserum. All antisera reacted equally well with twelve isolates ofC. sepedonicum obtained from Canada, California, Montana, Oregon, Washington, and Wisconsin. No cross-reactivity was observed when the anti-C. sepedonicum sera were tested against five otherCorynebacterium spp.and seven other species of plant pathogenic bacteria.

Studies of Introital Colonization in Women with Recurrent Urinary Infections. IV. The Role of Local Vaginal Antibodies

Immunoglobulins IgG and IgA were determined in 94 collections of vaginal fluid from 10 patients before, during and after 17 episodes of bacteriuria. The results were compared to 49 collections from 6 volunteer controls who had never had bacteriuria. The mean IgG concentration in patients (48 plus or minus 54mg. per cent) was no different from controls (50 plus or minus 33mg. per cent). IgA concentrations were 2.6 plus or minus 2.1 and 2.6 plus or minus 2.4mg. per cent, respectively. Bacterial agglutination titers in vaginal fluid to Escherichia coli colonizing the vaginal vestibule of the patients were no different (7.7 plus or minus 9.8, reciprocal tube titer) than the titers to the predominant Escherichia coli cultured from the rectum of the controls (9.7 plus or minus 5.0). Longitudinal studies in patients failed to show any relationship between vaginal colonization with changing serotypes of Escherichia coli and agglutination titers in vaginal fluid.

Clinical, Bacteriological, and Serological Observations of two Human Volunteers Following Ingestion of Escherichia coli

1. Two healthy, adult white, male volunteers ingested, respectively, 4 and 8 million organisms of E. colt serotype 0127: B8. 2. Symptoms of mild to moderate gastroenteritis appeared in both subjects 7 to 17 hours after ingestion of the organisms and persisted for 32 to 46 hours. 3. By daily rectal swab cultures, E. colt 0127:B8 organisms were recovered within 24 hours after ingestion and were found during 5 days in one and 12 days in the second volunteer. 4. Hemagglutinins were detected 3 days and bacterial agglutinins 5 days following ingestion. Antibody titers in both subjects rose rapidly, reaching peaks by the eighth to ninth day. Hemagglutination titers were consistently higher than bacterial agglutination titers. 5. Challenge ingestion of 8,000.000 organisms by one volunteer was followed by bacterial and serological response without appearance of clinical symptoms.