Passive Hemagglutination Titers Research Articles

Comparative efficacy of BAU-fowl cholera and DLS-fowl cholera vaccines in indigenous chicken

The present study was conducted to determine the immune response induced in indigenous chicken produced against BAU-FC and DLS-FC vaccines with their efficacy study against Pasteurella multocida. A total of forty (40) chickens were selected and divided into Group A (15), Group B (15) and Group C (10). Group A and B were vaccinated with BAU-FCV and DLS-FCV, respectively at the dose rate of 0.5 ml through SC at six weeks of age followed by boostering at 10 weeks of age while Group C was kept as unvaccinated control. Sera samples were collected after primary and booster vaccination and antibody titre was determined by Passive hemagglutination (PHA) test. The mean PHA titres recorded at 4 weeks after primary vaccination was 51.20 ± 7.84 in birds of group A and 38.40 ± 6.40 in birds of Group B. After booster vaccination, mean PHA titer was found 140.80 ± 31.35 at 16 weeks of age in case of BAU-FC vaccinated group and 115.20 ± 12.80 in case of DLS-FC vaccinated group. The mean PHA titer was 204.80 ± 31.35 and 179.20 ± 31.35 at 19 weeks of age in birds of BAU-FC and DLS-FC vaccinated group, respectively. Birds of all groups were challenged with virulent P. multocida at 17 weeks of age. It was observed that vaccinated chickens showed maximal resistance (100%) following challenge with virulent whereas unvaccinated control birds failed to resist the challenge infection. It can be assumed from the findings of present research work that both BAU-FCV and DLS-FCV are able to protect indigenous chicken from the outbreak of avian pasteurellosis and BAU-FV vaccine showed relatively higher immuno-protective titre than that of DLS-FC vaccine.Res. Agric., Livest. Fish.5(2): 193-199, August 2018

Immune response of fowl cholera vaccine produced at Bangladesh Agricultural University in farm level

The work was carried out to determine the immunogenicity of fowl cholera vaccine (FCV) produced by Livestock and Poultry Vaccine Research and Production Centre (LPVRPC) at Bangladesh Agricultural University (BAU). A total five hundred of sevenweek old Hy-sex chickens (both white and brown) were vaccinated @ 0.5 ml of 2.93×108 CFU through subcutaneous route in each selected groups such as A1, A2 and A3; and B1, B2 and B3. Booster dose was provided at 13 weeks of age in group A3 and B3. Group C was kept as unvaccinated/control. Postvaccination sera were collected at different time schedule from all the groups of birds and antibody against fowl cholera were determined by Passive haemagglutination (PHA) test. At 4 weeks of primary vaccination (11 weeks aged birds) the mean PHA titres of sera were 96.00±34.21 and 96.00±34.21in group A1 and B1 respectively. On the other hand mean PHA titres at 5-weeks following vaccination (12 weeks aged birds) were 88.00±33.12 and 96.00±34.21in group A2 and B2, respectively. After 4 weeks of booster vaccination the mean PHA titres were 104.00±33.12in A3 and 104.00±33.12 in B3 group. The mean PHA titres in chickens of unvaccinated control group C was <4±0.00.Fowl cholera vaccine prepared at LPVRPC induced a good level of immunity at the farm level and it was also demonstrated that booster (secondary) vaccination is essential to develop protective level of immunity.Res. Agric., Livest. Fish.2(1): 103-107, April 2015

Isolation and Identification of Pasteurella multocida from Chicken for the Preparation of Oil Adjuvanted Vaccine

The research work was performed for the isolation and identification of Pasteurella multocida from field cases, preparation of oil adjuvanted vaccine from isolated strain and determination of its efficacy. Samples were collected from suspected dead birds of three poultry farms of Bangladesh (Code name: M and R). The P. multocida isolates were Gram negative, non-motile, non- spore forming rod occurring singly or pairs and occasionally as chains or filaments. Biochemically P. multocida ferment basic sugar and consistently produced acid except from maltose and lactose. After isolation formalin killed oil adjuvanted Fowl cholera vaccine was prepared in Laboratory of the Department of Microbiology and Hygiene, BAU and this experimental vaccine (3.2x108 CFU/ml) was administered in nine weeks old White Leg Horn chickens at the different dose rate through intramuscular (IM) route in each selected group A (1ml alum precipitated vaccine), B (0.5ml alum precipitated vaccine), C (1ml oil adjuvanted vaccine) and D (0.5ml oil adjuvanted vaccine). Pre-vaccinated sera were collected from all groups of birds. The mean of Passive Hemagglutination (PHA) titers of post-vaccination were 51±17.8, 76.8±17, 89.6±17, and 115±17.81 in group A, B, C and D respectively which consist of 5 birds in each. The vaccine produced better immune response when boostering with the similar dose and route at 15 days after primary vaccination. The mean PHA titers were higher at group D than other groups after boostering. Challenge infection was conducted on all the vaccinated and control group (n=5) of birds after 15 days of vaccination which protect 93.75% of birds and the PHA titers from different groups analyzed to determine the protective capacity of vaccinated chickens against challenge exposure. It was demonstrated that experimental oil adjuvanted fowl cholera vaccine with 0.5ml dose produce higher immune response against challenge infection and found to be safe. Microbes and Health, June 2013, 2(1): 1-4DOI: http://dx.doi.org/10.3329/mh.v2i1.17253

Modification of the passive heamagglutination test for detection of <i>infectious bursal disease virus</i>

To modify the Passive Haemagglutination (PHA) test, a rapid test, used for qauntitative detection of viral antibodies, so that it can be used for determination of viral titres, dilutions of Infectious Bursal Disease Virus (IBDV) were used to sensitize the Red Blood Cells (RBCs) before reacting them with known IBD serum. Also, to improve sensitivity of the test, different RBC concentrations were used for the test. A standard IBDV gave positive PHA reaction upto its 1:2048 dilution. With different IBDV samples, positive PHA reactions occured upto dilutions, ranging from 1:16 to 1:4096. Different RBC concentrations gave different titres for same IBDV samples. With 0.6% and 0.2% RBC concentrations, mean PHA titres of IBDV samples increased from 454. 85 ± 315.32 to 2396.57 ± 489.55 (p < 0.05 ). It was concluded that PHA can be adopted for evaluation of viral titres. To improve sensitivity of the test, use of 0.2% RBC is recommended.

Immunopotency of novel oil adjuvant vaccines employing Pasteurella multocida biofilm and capsule enhanced organisms in ducklings

Pasteurella multocida-a gram-negative bacterium with a broad host range and ubiquitous distribution—is a serious problem in ducks, and the best method of control is vaccination with locally prevalent strains. The present study was conducted to compare the immunopotency of vaccines employing P. multocida biofilms and capsule enhanced organisms with ordinary broth grown organisms in 1-month-old ducklings, by measuring the humoral immune response and the protection conferred by each vaccine during homologous challenge with virulent organisms. Formalin inactivated oil adjuvant bacterin vaccines were prepared from Pasteurella multocida A: 1. They were grown in conventional broth, capsule enhancement medium, and under biofilm mode. Four different groups of birds were vaccinated with the respective vaccines by intramuscular route, and the immunopotency of the vaccines was assessed by employing passive haemagglutination (PHA) and homologous challenging with virulent organisms. The PHA titres obtained for the biofilm vaccine group on day 14 postvaccination were much higher than those for the other 2 groups. They also provided 10% more protection when challenged with 200 and 100 median "lethal dose" doses. The capsule enhanced vaccine and conventional bacterin revealed similar results.

Comparative Evaluation of Cell Culture-Adapted and Chicken Embryo-Adapted Fowl Pox Vaccine Strains

Two types of vaccines, chicken embryo adapted (VacCE) and cell culture adapted (VacCC), were tested for their efficacy to elicite the immune response in birds vaccinated at 2 and 8 wk of age. The cell-mediated immune response studied by blastogenesis assay showed that birds vaccinated at the second week of age by both VacCE and VacCC vaccines had significant increase in T-lymphocyte count at 21 days postvaccination (PV) and 7 days postchallenge (PC), whereas in birds vaccinated at 8 wk of age, a significant increase was seen at 21 days PV and 7 days PC with the VacCC vaccine. The rise in passive hemagglutination titers was observed up to 21 days PV and 7 days PC in birds vaccinated at 2 wk of age. However, only the birds vaccinated with VacCC at 8 wk of age showed rise in titers at days 21 PV and 7 PC. Birds were challenged 90 days PV by scarification on the thigh region, and the birds vaccinated with VacCC showed 90% and 70% protection when vaccinated at 2 and 8 wk, respectively. The birds vaccinated with VacCE showed only 60% and 20% protection at the corresponding levels, respectively.

Serological Evidence of Bovine Herpesviruses 1 and 2 in Asian Elephants

Antibodies were detected against bovine herpesviruses 1 (BHV 1) and 2 (BHV 2) in Asian elephants (Elephas maximus) using the passive hemagglutination (PHA) test. The study was conducted during May to December 1994 using sera collected from zoological gardens and national parks in India. Four (4%) of 109 elephant sera had PHA titers ranging from 1:8 to 1:32 against BHV 1. Twenty-five (23%) of the 109 elephant sera had PHA titers ranging from 1:8 to 1:64 against BHV 2. Asian elephants appear to be better reservoirs for herpesviruses which are serologically related to BHV 2.

Diagnosis of paracoccidioidomycosis by passive haemagglutination assay of antibody using a purified and specific antigen-gp43.

Sheep red blood cells, treated with glutaraldehyde and sensitized with a purified Paracoccidioides brasiliensis antigen of molecular mass 43 kDa (gp43), were used to detect antibodies to P. brasiliensis by passive haemagglutination (PHA). Sera from 50 patients with paracoccidioidomycosis had PHA titres of > 1/800, and had antibodies detectable by immunodiffusion while antibodies to gp43 were detectable by PHA in sera from patients with histoplasmosis, aspergillosis, candidiasis, Jorge Lobo's disease or healthy people, only at titres of < 1/100. Considering a cut-off at 1/200, the sensitivity and specificity of PHA for paracoccidioidomycosis was 100%. Thus, the PHA test could be an efficacious method for routine serological diagnosis of paracoccidioidomycosis, with results in 1 to 2 h.

Guinea pig model of immunologic asthma induced by inhalation of trimellitic anhydride.

We established a model of asthma induced by trimellitic anhydride (TMA) in guinea pigs and assessed the role of sensitization in the development of their bronchial hyperresponsiveness, and relationship between bronchial responsiveness and bronchial inflammation. Fourteen guinea pigs (sensitized group) were administered 1 mg/0.5 ml of trimellity 36-bovine serum albumin intramuscularly and 0.5 ml of complete Freund adjuvant on Day 1 as the priming dose. Booster doses were repeated on Day 15. By Day 28, all of the sensitized animals showed a high passive hemagglutination titer against trimellityl 14-ovalbumin. On Day 29, they were challenged by an inhalation of TMA (150 mg/m3) for 30 min, and respiratory resistance (Rrs) was monitored by the oscillation method. In all sensitized animals, Rrs increased immediately upon challenge and returned to baseline within 6 h. The bronchial reactivity to acetylcholine (Ach), measured 6 h after TMA challenge in the sensitized animals, increased significantly (p < 0.01) compared with that measured 24 h before challenge; that measured 24 h later was not different from that before challenge. There was also a significant difference (p < 0.01) in the number of eosinophils in the lamina propria and the epithelium 6 and 24 h after the challenge inhalation in the sensitized group. The increased airway responsiveness to Ach in the sensitized animals was correlated with an increase in the number of eosinophils in the lamina propria and the epithelium. These observations suggest that humoral antibody and eosinophils are involved in the pathogenesis of TMA-induced asthma.

Passive hemagglutination test for detection of antibodies against Taylorella (Haemophilus) equigenitalis in Sera of Mares

The passive hemagglutination (PHA) test was improved to enable the detection of antibodies to Taylorella (Haemophilus) equigenitalis in the sera of mares. Horse red blood cells (RBC) fixed with glutaraldehyde were compared with similarly treated RBC of a cow, pig and sheep for the PHA test. The horse RBC were superior to those of the other animals tested in detecting mares affected with contagious equine metritis (CEM). A PHA test using these cells as indicator and an antigen prepared from T. equigenitalis by sonication following treatment with hyaluronidase was the most satisfactory in terms of sensitivity and specificity. None of the 156 serum samples from clinically healthy mares without a history of contact with T. equigenitalis-infected stallions or mares showed PHA titers >1:32 and only a few samples (7.1%) showed PHA titers of 1:32. Four of the 50 serum samples from mares affected with CEM showed PHA titers of 1:32, while most of the samples (92.0%) showed PHA titers >1:32. The glutaraldehyde-fixed horse RBC sensitized with the antigen had the advantage of being reproducible for at least 7 months when preserved at 4°C.

Serological detection of Edwardsiella ictaluri Hawke lipopolysaccharide antibody in serum of channel catfish, Ictalurus punctatus Rafinesque

Abstract. Bacterial agglutination, passive haemagglutination, complement‐dependent passive haemolysis, indirect immunofluorescence, agar gel immunodiffusion and agglutination with fractions of immunized fish serum were compared for detecting humoral antibody to the lipopolysacchande (LPS) of Edwardsiella icialuri Hawke in channel catfish. Bacterial agglutination titres averaged 1: 672; passive haemagglutination titres averaged 1: 1152; and complement‐dependent haemolysis titres averaged 1: 2360. Serum from non‐vaccinated fish ranged from 0 to 1:32. Indirect fluorescence and immunodiffusion demonstrated positive reactions to the LPS antibody. Fractionation of immune sera produced three fractions, one of which strongly haemagglutinated E. ictaluri but the other two did not. All six serological techniques were sensitive to E. ictaluri LPS antibody.

Antigenic stimulation with proteins of cow's milk via the oral route in guinea pigs and rats. 2. Antibodies to beta-lactoglobulin secreted into the alimentary canal and serum.

The intestinal contents of guinea pigs and rats on a milk-drinking diet were examined for mucosal-secreted antibody to beta-lactoglobulin. Assay of the immunoglobulin content of duodenum, jejunum and ileum of the guinea pig gave geometric means of 70, 90 and 95 micrograms/ml. In the rat, where isotype-specific antiglobulin reagents were available, similar figures were obtained but it could be shown that the vast majority was IgA. Passive haemagglutination titres up to 1,000 of secreted anti-beta-lactoglobulin antibody (resistant to the endogenous proteases in the intestinal contents) were measured in guinea pigs. Fifteen days after cessation of milk-drinking, the antibody had dropped to undetectable levels. No such antibody could be shown in rats. Rats also failed to develop detectable serum antibody whereas, the guinea pigs had passive haemagglutination titres in the order of 16,000.

Passive hemagglutination test for enteric fever.

A passive hemagglutination (PHA) test for serodiagnosis of enteric fever was developed by sensitizing glutaraldehyde-preserved erythrocytes with lipopolysaccharide from Salmonella serogroups A, B, C, and D singly or simultaneously. The lipopolysaccharide-sensitized erythrocytes were tested with sera from 200 blood donors, 100 patients whose hemoculture was positive for Salmonella species, and 10 patients septicemic for other members of the family Enterobacteriaceae. The PHA test was positive in 90% of 28 acute-phase serum samples from patients with enteric fever from one hospital and in 93% of 72 acute-phase serum samples from another hospital. It was also positive in 100 and 60% of early- and late-convalescent-phase sera, respectively. The PHA test was negative in all patients septicemic for other members of the Enterobacteriaceae. Absorption of sera from patients with enteric fever with lipopolysaccharide from other members of the Enterobacteriaceae did not reduce PHA titers, indicating the specificity of the PHA test. Simultaneous sensitization with lipopolysaccharide from Salmonella serogroups A, B, C, and D was useful as a screening test in a limited trial with 28 acute-phase sera, 10 early-convalescent-phase sera, and 17 late-convalescent-phase sera. The PHA test is indeed a simple, sensitive, specific, and rapid test supplementing hemoculture in laboratory diagnosis of enteric fever.

Detection of an Antigen (AN6520), Possibly Related to Non‐A, Non‐B Hepatitis, by Monoclonal Antibodies. I

The antibody to AN6520 antigen, which was isolated from the liver of a patient with non-A, non-B hepatitis (NANBH), has been detected frequently in convalescent sera from patients with NANBH by the passive hemagglutination (PHA) test. In a further study, we established hybridoma cells secreting antibodies against AN6520 antigen and obtained ascitic fluids with PHA titers ranging from 1:10(5) to 1:10(7). In immunodiffusion with AN6520 antigen, all monoclonal antibodies were found to form an identical precipitin line. These lines were also identical to those formed by rabbit antiserum against AN6520 antigen and by convalescent sera from patients with NANBH. With one of the monoclonal antibodies, 1-F12, solid-phase radioimmunoassay (SP-RIA) for detecting AN6520 antigen was developed as well as blocking RIA for anti-AN6520 antibody detection. The antigen assay was 50 times more sensitive than the reverse passive hemagglutination (R-PHA) test, with a sensitivity threshold of the 1 ng/ml of antigen solution; the antibody assay was 10 times more sensitive than PHA. The results with this blocking RIA were mostly in agreement with the data obtained by PHA. Furthermore, the antigen in human sera, which had never been detected by R-PHA test, could be detected by SP-RIA.

Single-serum diagnosis of rubella by combined use of the hemagglutination inhibition and passive hemagglutination tests.

We explored the feasibility of using the passive hemagglutination (PHA) test in combination with the hemagglutination inhibition (HI) test for the single-serum diagnosis of rubella. We found by sedimentation analysis of the serum that early 7S antibody produced within 1 month after the onset of the rash had high HI but much lower PHA titers, whereas the PHA titers of antibody produced later were slightly higher than the HI titers. (19S antibody in the early serum had some PHA activity.) This disparity between the HI and PHA activities of the early 7S antibody could be used for the routine diagnosis of rubella. A comparison of the HI titers of the test serum with the PHA titers of mercaptoethanol-treated serum constitutes a simple method for determining whether the serum sample was taken shortly or remotely after the infection.

Production of Vibrio cholerae O1 and non-O1 typing sera in rabbits immunized with polysaccharide-protein carrier conjugates.

Two systems are currently used to serologically type Vibrio cholerae O1 and non-O1 isolates. Antiserovar-serotype serum in the Smith system is produced in rabbits immunized with live whole-cell vaccines, and that in the Sakazaki system is produced in rabbits immunized with heat-killed vaccines. In neither system is the serovar-serotype-specific antigen clearly defined. During the course of a serological survey, ca. 10% of more than 2,500 V. cholerae isolates examined agglutinated in the optimal dilutions of two, three, or four different anti-serovar sera prepared by the methods of Sakazaki. An occasional isolate agglutinated in both anti-O1 and non-O1 sera. Lipopolysaccharide was extracted from eight of these possible multiple serovars, coated onto rabbit erythrocytes, and retested in these same antisera by passive hemagglutination. With one exception the lipopolysaccharide-rabbit erythrocytes were now agglutinated in a single antiserum. Antipolysaccharide sera were produced in rabbits immunized with the polysaccharide moiety extracted from eight non-O1 and two O1 vaccine strains conjugated to bovine gamma globulin protein carrier. The antipolysaccharide sera showed passive hemagglutination titers versus lipopolysaccharide-rabbit erythrocytes comparable to those achieved in antisera from rabbits immunized with heat-killed whole-cell vaccines. In the slide agglutination test antipolysaccharide sera serologically discriminated between two O1 isolates that were previously agglutinated in both anti-O1 and anti-non-O1 whole-cell sera. It is recommended that serological types or varieties of V. cholerae non-O1 be based upon serologically recognizable differences in lipopolysaccharide-associated antigens as are antigens A, B, and C in the O1 group.

Immunoresponse of eel against Edwardsiella tarda antigens.

From Edwardsiella tarda EF-1 strain, three antigens were obtained: lipopolysaccharide (LPS), culture filtrate and formalin killed whole cells. Eel were injected intramuscularly with the antigens twice at an interval of one week. The agglutination and passive hemagglutination titers in serum were determined three weeks after the second immunization. In the another experiment, eel were challenged by intramuscular injection with live bacteria three weeks after the second immunization and the agglutination titer was determined in the serum of the surviving eels. Antigenic relationships between the LPS and the culture filtrate were examined by OUCHTERLONY method and the immunoelectrophoresis.All the immunized eel showed an increase in titer to the three antigens, but a large increase in the titer was not observed. The highest survival rate was recorded in challenged eel immunized with LPS, which indicated the LPS role may correlate with the protective effect of the vaccine. Agglutination titer increased significantly in the serum of the immunized eel, but no correlation between survival rate and agglutination titer was observed among the immunized groups. The results suggest that the agglutination titer using whole cell antigen does not correlate with the level of the protection observed in eels. The increase in the titer with whole cell antigen was considered to be a function of an antigen detected in the LPS and the culture filtrate.

A rapid standardized enzyme immunoassay for autoantibodies to thyroglobulin.

A rapid, specific, standardized, objective and sensitive quantitative enzyme immunoassay (ELISA) procedure has been developed for the detection of human autoantibodies to thyroglobulin. It involves three ten minute incubations, with the positive control serum adjusted to yield an absorbance of approximately 1.00. The results are reported as 'percentage of positive control' absorbance. The specificity of the reactions for thyroglobulin autoantibodies was supported by the good correlation found between the ELISA values obtained and the passive hemagglutination (PHA) titers of 348 sera from 333 patients suspected of having thyroid disturbances, as well as by antigen absorption experiments. Rheumatoid factor did not interfere with the assay. An appreciable proportion of passive hemagglutination-negative sera were confirmably positive for thyroglobulin autoantibodies by the ELISA method.

Use of combined immunization routes in induction of a salivary immunoglobulin A response to Streptococcus mutans in Macaca fascicularis monkeys.

Various combinations of immunization routes were examined for ability to elicit or prolong (or both) a salivary secretory immunoglobulin A response to Streptococcus mutans strain Ingbritt (serotype c) in Macaca fascicularis monkeys. Intraductal (i.d.), per os (p.o.), and subcutaneous (s.c.) routes were utilized. Four groups of three to five monkeys each were immunized by the following schedules: group I--p.o., s.c., i.d.; group II--i.d., p.o., i.d.; group III--s.c., p.o., i.d.; and group IV--control. Immune responses in the serum and parotid fluid were quantitated by using passive hemagglutination assays with purified serotype-specific polysaccharide and by indirect immunofluorescent staining assays. Both s.c. and i.d., but not p.o., routes resulted in detectable serum antibody responses. Only i.d. immunization resulted in a measurable salivary response. Indirect immunofluorescent staining revealed specific secretory immunoglobulin A antibodies in the parotid fluid which correlated with passive hemagglutination titers. The p.o. procedures used in this study did not result either in a prolonged immune response or in measurable tolerance related to the humoral or secretory immune system.

Hemagglutination titer of gamma-globulin preparations for intravenous use against OEP (original endotoxin protein), protease and elastase of Pseudomonas aeruginosa (author's transl)

Using formalinized sheep erythrocytes treated with tannic acid and coated with OEP (protein moiety of endotoxin), protease and elastase of Pseudomonas aeruginosa, passive hemagglutination titer was measured on three kinds of gamma-globulin preparations (13 lots from each maker).67%(26/39) of gamma-globuin preparations had detectable HA antibody against OEP. 97%(38/39) had HA antibody against protease, and 92%(33/36) against elastase. No significant difference was found between mean value of OEP-HA of pepsin treated and polyethyleneglycol (PEG) treated preparations. Sulfonated preparations had a tendency to have a low OEP-HA and the difference was significant (p<0.05) compared with pepsin treated preparations. In the case of protease-HA, pepsin treated treated preparation had the highest mean value and sulfonated preparation had lowest mean value. The difference was significant. No difference in elastase HA was noted among all three gamma-globulin preparations.