Passive Hemagglutination Research Articles

Psychoimmunomodulatory Effect of Phenotropil in Animals with Immune Stress

We studied the effect of phenotropil (25 mg/kg intraperitoneally, 5 days) on the immune and psychoemotional state of Wistar rats with LPS-induced immune stress. Hyperactivity of the immune system in animals after treatment with Pseudomonas aeruginosa LPS (100 μg/kg intraperitoneally, 3 days) manifested in a significant increase in the delayed-type hypersensitivity index, antibody titer in the reaction of passive hemagglutination, and phagocytic activity of peripheral blood neutrophils. Locomotor, orientation, and exploratory activities were reduced, while anxiety increased in animals with immune stress. Phenotropil exhibited the psychoimmunomodulatory effect under these conditions, which manifested in prevention of anxiety and fear response, increase in horizontal locomotion and exploratory behavior, and improvement of immunoreactivity.

Association of anti-IgA antibodies with adverse reactions to γ-globulin infusion

Association of anti-IgA antibodies with adverse reactions to γ-globulin infusion

Establishment of a cell line panel as an alternative source of platelet antigens for a screening assay of anti‐human platelet antibodies

A panel of platelets expressing various human platelet antigens (HPAs) for a platelet antibody screening assay is difficult to prepare because some antigens are rarely expressed. Therefore, an alternative method without using platelets would be helpful in detecting HPA antibodies. This study describes the establishment of cell lines that stably express specific HPAs and their application for detecting specific antibodies. Wild-type β3, HPA-1b, -6b, -7b and -7 variant cDNA as well as wild-type αIIb and HPA-3b cDNA were individually co-transduced with wild-type αIIb and β3 cDNA in the K562 cell line. We performed an immunobead monoclonal antibody immobilisation of platelet antigens (MAIPA) assay to evaluate this cell line panel for antibody detection using identified sera containing HPA antibodies, whose specificities had been determined by the mixed passive haemagglutination test. Of the 12 sera containing HPA-1a (n = 2), HPA-3a (n = 6), HPA-6b (n = 3) or HPA-7 variant (n = 1) antibodies, all antibodies were detected and determined by our new method, except for two HPA-3a antibodies. One of the two antibodies was also negative for conventional platelet MAIPA, suggesting that the cell line panel might be used as an alternative source of platelet antigens in the MAIPA assay.

ASSESSMENT OF A RECOMBINANT F1-V FUSION PROTEIN VACCINE INTENDED TO PROTECT CANADA LYNX (LYNX CANADENSIS) FROM PLAGUE

As part of an ongoing restoration program in Colorado, USA, we evaluated adverse reactions and seroconversion in captive Canada lynx (Lynx canadensis) after vaccination with a recombinant F1-V fusion protein vaccine against Yersinia pestis, the bacterium that causes plague. Ten adult female lynx received the F1-V vaccine; 10 source- and age-matched lynx remained unvaccinated as controls. All of the vaccinated and control lynx remained apparently healthy throughout the confinement period. We observed no evidence of injection site or systemic reactions to the F1-V vaccine. Among vaccinated lynx, differences in log(10) reciprocal antibody titers measured in sera collected before and after vaccination (two doses) ranged from 1.2 to 5.2 for anti-F1 antibodies and from 0.6 to 5.2 for anti-V antibodies; titers in unvaccinated lynx did not change appreciably over the course of confinement prior to release, and thus differences in anti-F1 (P=0.003) and anti-V (P=0.0005) titers were greater among vaccinated lynx than among controls. Although our findings suggest that the F1-V fusion protein vaccine evaluated here is likely to stimulate antibody responses that may help protect Canada lynx from plague, we observed no apparent differences in survival between vaccinated and unvaccinated subject animals. Retrospectively, 22 of 50 (44%; 95% confidence interval 29-59%) unvaccinated lynx captured or recaptured in Colorado during 2000-08 had passive hemagglutination antibody titers >1:16, consistent with exposure to Y. pestis; paired pre- and postrelease titers available for eight of these animals showed titer increases similar in magnitude to those seen in response to vaccination, suggesting at least some lynx may naturally acquire immunity to plague in Colorado habitats.

Evaluation of a fecal occult blood test with reverse passive hemagglutination for colorectal neoplasm screening.

To evaluate the one and three sampling reverse passive hemagglutination fecal occult blood test (RPHA FOBT) for colorectal neoplasm screening. A group of 3034 individuals with histories of colorectal polyps and/or ulcers were screened for colorectal cancer. Three day fecal samples were collected and 60 cm fiberoptic colonoscopy was conducted for each subject. The fecal samples were tested for occult blood with the RPHA method and the endoscopic and histopathological diagnoses were used as standard reference for evaluation. The sensitivity, specificity and positive and negative predictive values of different samplings were compared. About 521 cases of colorectal neoplasms were detected, including 12 cases of colorectal cancer and 509 cases of polyps. Results showed that the mean sensitivity of one sampling RPHA FOBT for colorectal neoplasm was only 13.2%, the specificity was 90.3% and the positive and negative predictive values were 21.3% and 83.4%, respectively; while for the three sampling, taking one positivity as positive, the sensitivity increased to 22.0%, the specificity decreased to 81.6% and the positive and negative predictive values were 19.7% and 83.6%, respectively. A single RPHA FOBT seems to be less sensitive for screening for colorectal neoplasms. Since it is convenient and economical, RPHA FOBT remains the most practical procedure for detection of early colorectal cancer and polyps if it is combined with other screening methods.

B型肝炎感染対策の実際

The hepatitis B virus (HBV) is a serious concern for people at high risk of exposure to blood in their work because it is very infectious. While infection can be prevented by acquiring antibodies from hepatitis B vaccine, there are low responders and non-responders to the vaccine.We examined 2 possible methods of overcoming this problem: performing an additional vaccination with a different type of vaccine and carrying out 2 additional vaccinations at monthly intervals. Regarding the former method, an additional vaccination with a different vaccine type was carried out for a subject who could acquire no antibodies after undergoing 2 series of 6 vaccinations and who had not had any antibodies to hepatitis B for about 10 years. One month later, the passive hemagglutination test (PHA test) showed that the subject had obtained a 512-fold increase in the number of antibodies. As for the latter method, 3 of 5 non-responders acquired antibodies. Also, 5 of 6 subjects who were less responsive to the vaccine acquired antibodies and maintained them for at least 3 years.We intend to improve immunization coverage by using the methods examined in this study.

Ovarian hydrobursitis in female camels ( Camelus dromedaries): Biochemical, bacterial and protozoal evaluation

The aim of this study was to evaluate female camels affected with ovarian hydrobursitis (n = 31) for hematological and biochemical findings and for bacterial and protozoal infections. Blood samples were obtained and surgical ablation of the affected bursa was performed. Bursal fluid, follicular fluid, and serum were subjected to hormonal and biochemical analyses. Bursal fluids were cultured and colonies were identified using BioMérieux Vitek two compact system. Passive haemagglutination test was used for detection of Trypanosoma evansi. Indirect ELISA technique was carried out for detection of anti-Hydatid cysts anti-bodies. Neutrophilia was found in the affected animals (P = 0.01) with tendencies for monocytosis (P = 0.06) and eosinophelia (P = 0.05). Bursal fluid had a tendency for high estradiol-17 β concentration compared to blood serum (P = 0.07). Progesterone and cholesterol concentrations were similar in bursal fluid, follicular fluid and serum. Total protein, phosphorus, and magnesium concentrations were greater (P < 0.05) in the bursal fluid than in serum. Oligella urethralis, Alloiococcus otitis, Granulicatella adicens, Escherichia coli, Sphingobacterium thalpophilum, Streptococcus sanguinis, Aeromonas salmonicida, Pseudomonas stutzeri, Staphylococcus warneri, Staphylococcus hominis, and Rhizobium radiobacter were isolated from 46.7% of bursal fluids. T. evansi was positive in 9.7% of cases . None were positive for hydatid cyst. Accordingly, we suggest that the ovarian hydrobursitis syndrome is initially an inflammatory process and the accumulated bursal fluid is partially originated from follicular fluid.

Antigenic Drift of Orientia tsutsugamushi in South Korea as Identified by the Sequence Analysis of a 56-kDa Protein-Encoding Gene

This study was performed to determine the nationwide antigenic diversity of Orientia tsutsugamushi in South Korea. Sequence analysis was performed around variable domains I and II of a 56-kDa protein-encoding gene. We used eschar to overcome the disadvantages of conventional serotyping. The serological passive hemagglutination assay (PHA) was assessed based on the genotyping results. We analyzed 153 isolates from scrub typhus patients in major endemic areas and found that Boryong was the major strain (68.6%). New strains were also identified: Taguchi (19.6%), Kanda/Kawasaki (9.2%), and UAP7 (1.3%). PHA yielded significantly fewer positive results among Kawasaki strains (P < 0.001), which are not included in the PHA antigen panel. In South Korea, Boryong was still the predominant strain, but the sequence analysis identified new changes in minor strains (30.1%). This antigenic drift had a negative effect on the PHA results. Periodic surveillance of the contemporary strains using sequence analysis is needed.

Rinoneumonitis equina en Chile (1969 -1992)

In this general article is presented the situation of equine rhinopneumonitis in Chile from 1969, when a long and heavy outbreak of abortion occurred at the Central Zone of the country, causing serious losses in thoroughbred. After a program of immunization iniciated in 1974 by using a modified live vaccine (Rhinomune), equine rhinopneumonitis decreased gradually. Equine herpes virus type 1 was isolated from liver of aborted fetuses and nasal or ocular discharges of sick adult horses. In 1984 the virus was isolated again from aborted fetuses belong to Victoria, IX Region of Chile; this is the only case of equine rhinopneumonitis described at the south of the country. Moreover, different studies were done about the use of complement fixation test, viral seroneutralization, passive haemag glutination test and direct fluorescent technique to diagnose equine rhinopneumonitis; adaptation of the virus in guinea pigs and post vaccinal response to modified and inactivated vaccines against this disease. Finally it is discussed the current situation of equine rhinopneumonitis in Chile. Key words: Virus, equine rhinopneumonitis, horses. Palabras claves: Virus, rinoneumonitis equina, quinos.

Allergic transfusion reactions from blood components donated by IgA‐deficient donors with and without anti‐IgA: a comparative retrospective study

IgA deficiency is common (1/500) and up to 40% of affected individuals will develop anti-IgA. A few studies suggested that passive transfusion of anti-IgA was not associated with an increased risk of allergic reactions. This study was designed to assess the safety of transfusing blood components containing anti-IgA. IgA-deficient blood donors with and without anti-IgA were identified from Héma-Québec's (HQ) computerized database. IgA deficiency was confirmed by an ELISA method and the presence of anti-IgA by a passive hemagglutination assay. Blood donations from IgA-deficient donors issued to hospitals between March 1999 and December 2004 were retrieved. Medical charts of recipients were reviewed for the occurrence of a suspected transfusion reaction. Presence and nature of transfusion reactions were assessed blindly by an adjudicating committee. A total of 323 IgA-deficient blood products were issued by HQ to 55 hospitals. Of these, 48 agreed to participate [315 blood products (97.5%)]. A total of 272 products were transfused: 174 contained anti-IgA, and 98 did not. Only two minor allergic reactions occurred in each group. Incidence of allergic reactions was 1.15% in the anti-IgA group and 2.04% in the group without anti-IgA (P = 0.91). There was no anaphylactic reaction in either group. This study indicates that the proportion of allergic reactions does not appear to be greater in recipients of blood components containing anti-IgA compared to recipients of non-anti-IgA-containing components. Allowing donations from IgA-deficient donors with anti-IgA may therefore be contemplated.

External Quality Assessment for the Determination of Diphtheria Antitoxin in Human Serum

Accurate determination of diphtheria toxin antibodies is of value in determining the rates of immunity within broad populations or the immune status of individuals who may be at risk of infection, by assessing responses to vaccination and immunization schedule efficacy. Here we report the results of an external quality assessment (EQA) study for diphtheria serology, performed within the dedicated surveillance network DIPNET. Twelve national laboratories from 11 European countries participated by testing a standard panel of 150 sera using their current routine method: Vero cell neutralization test (NT), double-antigen enzyme-linked immunosorbent assay (ELISA; DAE), dual double-antigen time-resolved fluorescence immunoassay (dDA-DELFIA), passive hemagglutination assay (PHA), toxin binding inhibition assay (ToBI), and in-house or commercial ELISAs. The objective of the study was not to identify the best assay, as the advantages and drawbacks of methods used were known, but to verify if laboratories using their routine method would have categorized (as negative, equivocal, or positive) a serum sample in the same way. The performance of each laboratory was determined by comparing its results on a quantitative and qualitative basis to NT results from a single reference laboratory, as this test is considered the in vitro "gold standard." The performance of laboratories using NT was generally very good, while the laboratories' performance using other in vitro methods was variable. Laboratories using ELISA and PHA performed less well than those using DAE, dDA-DELFIA, or ToBI. EQA is important for both laboratories that use in vitro nonstandardized methods and those that use commercial ELISA kits.

Screening of Canadian Blood Services donors for severe immunoglobulin A deficiency

Immunoglobulin A (IgA)-deficient patients with antibodies to IgA require transfusions with IgA-deficient blood components to either avoid or reduce the frequency of serious adverse reactions. To supply compatible blood components for these individuals, the Canadian Blood Services (CBS) National Testing Laboratory must initially screen and subsequently identify, after confirmatory testing at the American Red Cross (ARC), donors severely deficient in IgA (<0.05 mg/dL). The Ouchterlony double immunodiffusion assay was used as an initial screen at CBS to identify IgA-deficient donors (test sensitivity 2-4 mg/dL). Sample aliquots from these donors were then sent to the ARC for confirmatory testing using an enzyme-linked immunosorbent assay method for severe IgA deficiency (<0.05 mg/dL) and a passive hemagglutination assay to detect anti-IgA. From November 2007 to December 2008, of 54,594 samples screened initially at CBS there were 137 samples (0.251%) identified as possibly IgA deficient. Of these 137, there were 100 reports returned from ARC confirming severe IgA deficiency in 65 donors (25 female, 40 male) without detectable anti-IgA and in 35 donors (18 female, 17 male) with anti-IgA. The remaining 37 donors had IgA levels of more than 0.05 mg/dL. Results from the ARC confirmed a frequency of 1 in 546 in the CBS' blood donor population for severe IgA deficiency (<0.05 mg/dL), 1 in 840 for those without anti-IgA, and 1 in 1560 for those with antibody. Donors repeatedly confirmed as severely IgA deficient without anti-IgA were considered eligible for the CBS IgA-deficient donor registry program.

The Isolated and Purified β-lactamase from Local Isolate of Staphylococcus aureus

β-lactamase was isolated from a local isolate of Staph. aureus, which elicited its resistance to penicillin–G, and rapid β-lactamase production. β-lactamase has been purified by using gel-filtration chromatography in Sephadex G–100 column. The molecular weight of purified β-lactamase was estimated by SDS-poly acrylamide gel electrophoresis and shown one band protein with molecular weight of 30 kDa. The antiserum was prepared for purified β-lactamase in rabbits, the measurements of antibodies titer were done by using passive haemagglutination test and it equaled to 40960, which indicated the immunogenicity of purified β-lactamase. The neutralization of β-lactamase activity by antiserum was performed in vitro, so Staph. aureus isolates regained their sensitivity to penicillin–G.

Pesquisa de autoanticorpos em pacientes com artrite psoriásica sob terapia anti-TNFα

Anti-TNFα therapy has been effective in the treatment of patients with refractory psoriatic arthritis (PSA). However, the risk of developing autoantibodies commonly found in rheumatic diseases in PSA patients undergoing this therapy is not clear. To evaluate the induction of specific autoantibodies after anti-TNFα therapy in PSA patients. Serum samples from 23 PSA patients (women: 61%, age: 45.04 ± 12.68 years, polyarticular: 69.6%, disease duration: 13.3 ± 7.7 years, infliximab: 82.60%) obtained immediately before (baseline) and approximately one year after the introduction of anti-TNF therapy (last sample) (385 ± 131.45 days), were analyzed. The analysis included detection of antinuclear antibodies (ANA) and anti-dsDNA antibodies (indirect immunofluorescence on Hep-2 cells and Crithidia luciliae, respectively); anti-RNP and anti-Sm (passive hemagglutination); and anti-Ro/ SS-A and/or anti-La/SS-B, anti-chromatin, anti-histones, anti-citrullinated peptide (CCP), and anti-cardiolipin (ELISA) antibodies. At baseline, ANA was positive in 47.8% of patients, with predominance of homogeneous nuclear pattern (81.8%). All baseline serum samples were negative for rheumatoid factor and antibodies to cardiolipin, RNP, Sm, Ro/SS-A, anti-La/SS-B, anti-histone, and anti-dsDNA antibodies, while two patients were positive for anti-chromatin and one for anti-CCP. All ANA-positive samples at baseline, except for one, remained positive after the introduction of anti-TNF therapy; however, de novo ANA reactivity was observed in four originally negative patients (33.3%). Anti-Ro/SS-A, La/SS-B, cardiolipin, histones, dsDNA, and rheumatoid factor antibodies remained negative in all final serum samples tested, and anti-chromatin positivity was detected in three other patients. Our findings have shown that anti-TNF therapy induced ANA positivity in one third of PSA patients. The concomitant use of methotrexate did not interfere with this finding. In addition, all serum samples were systematically negative for specific rheumatic autoantibodies tested after the introduction of the biological treatment.

A serosurvey of Orientia tsutsugamushi from patients with scrub typhus

AbstractMany countries where scrub typhus is endemic use their own cutoff values for antibody titres to differentiate between cured cases and current infections. To establish an antibody titre cutoff value, one needs to investigate the seroprevalence in endemic areas, and the duration of the increase in titre after complete cure. We conducted a follow-up study of anti-Orientia tsutsugamushi antibody titres using indirect immunofluorescence assays (IFA) and passive haemagglutination assays (PHA) in patients with scrub typhus. After the onset of symptoms, IgM antibody titres increased gradually over 2–3 weeks, peaked at about 4 weeks, and started to decrease rapidly between 4 and 5 weeks. At 1-year follow-up, the median IgM value was 1:10. Out of 77 patients who were tested at that time, 36 (47%) had IgM titres ≥1:20, and none had titres exceeding 1:80. Over the first 2 weeks, IgG antibody titres increased sharply, peaked at about 4 weeks and decreased rather gradually thereafter, with a median titre of 1:128 maintained up to the 18th month. At 1-year follow-up, five out of 77 patients (6.5%) had titres ≥1:1,024 and 57% had titres ≥1:128. Based on these results, a cutoff value of ≥1:160 for IgM antibody should differentiate between previous and current infections in endemic areas such as Korea and Japan, where scrub typhus occurs mainly in the autumn.

The first case of alloantibody against human platelet antigen‐15b in Japan: possible alloimmunization by a hydatidiform mole

The involvement of the human platelet antigen (HPA)-15 system in neonatal alloimmune thrombocytopenia (NAIT) has been reported in various populations, but not in the Japanese population. In Japan, the mixed passive hemagglutination assay (MPHA) is used for detection of HPA alloantibodies. However, most of the reported cases of HPA-15 incompatibility are based on the monoclonal antibody immobilization of platelet antigen (MAIPA) assay or immunoprecipitation; thus there is a possibility that HPA-15 alloantibodies are not efficiently detected by the MPHA, and currently, the causative antibody is not detectable in approximately half of the suspected NAIT cases in Japan. We examined the sera of mothers from NAIT cases, previously with undetected HPA antibodies by MPHA, using the MAIPA technique. Sera from 90 mothers of suspected NAIT were tested by MAIPA for the presence of anti-HPA-15 alloantibodies. Anti-HPA-15b was detected in one case. This case was a mother in the first pregnancy diagnosed as hydatid mole-coexisting fetus, and the baby was born with suspected NAIT. The familial analysis revealed compatibility of HPA-15 genotype between the mother and the baby (both HPA-15a/a), but incompatibility with the paternal one (HPA-15a/b). The hydatid mole's tissue was genotyped as HPA-15b positive. Besides anti-HPA-15b, maternal sera contain strong HLA Class I antibody Here we reported the first case of anti-HPA-15 in Japan. Alloimmunization against the hydatid mole seems to be responsible for the production of HPA-15b alloantibody. This antibody, however, did not apparently involve in the development of NAIT of the newborn, the coexisting anti-HLA Class I being the possible cause.

Comparison of Nine Different Qualitative HBsAg Assay Kits

Qualitative hepatitis B surface antigen (HBsAg) assay kits are still commonly used in Korea where hepatitis B virus (HBV) infection is endemic. The accurate determination of HBsAg plays a crucial role in the diagnosis and prevention of HBV infection, especially in endemic areas. The aim of this study was to compare the detection sensitivities of 9 qualitative HBsAg assay kits. Seven pooled sera with HBsAg concentration ranging from 0.14 IU/mL to 29.96 IU/mL were prepared. The HBsAg concentration of each pooled serum was determined by a quantitative HBsAg assay, Architect HBsAg (Abbott Laboratories, Ireland). The fully automated immunoassay kits included Elecsys HBsAg (Roche Diagnostics, Germany) and Immulite 2000 HBsAg (DPC, USA) and the rapid tests included 5 immunochromatographic assay (ICA) kits and 2 reverse passive hemagglutination assay (RPHA) kits. Elecsys HBsAg (Roche Diagnostics) showed positive result in pooled serum with HBsAg concentration of 0.14 IU/mL, but Immulite 2000 HBsAg (DPC) showed negative result in the same concentration. Although ICA kits showed variable results among different assay kits, all of them showed negative results in pooled sera with HBsAg concentration of < or = 1.89 IU/mL. Two RPHA kits showed negative results in pooled sera with HBsAg concentration of < or = 7.98 IU/mL. Although ICAs were more sensitive than RPHAs, they had variable sensitivities for HBsAg and were less sensitive than the automated immunoassay kits. Therefore, ICAs and RPHAs should be used with caution in the screening tests for HBsAg and their sensitivities need to be improved.

Detection of Rotavirus in Stools by Means of Reversed Passive Haemagglutination (R-PHA)

Detection of Rotavirus in Stools by Means of Reversed Passive Haemagglutination (R-PHA)

Epizootiology of lumpy skin disease outbreak in cattle in middle of Egypt, 2006

The current investigation studied an outbreak of lumpy skin disease of cattle in Beni-Suef and Al-Fayium governorates from March up to September 2006. Epidemiological data over a total of 5500 cattle from all ages, breeds and sexes were investigated. Prevalence of lumpy skin antibodies was screened by enzyme-linked immunosorbent assay (ELISA) that revealed high exposure rates; 57% and 51.42% in Beni-Suef and Al Fayium governorates respectively Virus isolation was conducted on chorioallantoic membrane (CAM) of specific pathogen free embryonated chicken egg (SPF-ECE) and MDBK cell culture. The virus identity was confirmed by passive haemagglutination and polymerase chain reaction (PCR) on the infected CAM and MDBK cell culture. Experimental infection of rabbits was successful, demonstrating their possible roles in the epidemiological process of the disease.

Effect of uses of Probiotic prepared from Lactobacillus acidophilus Bacteria as immunostimulator in lambs vaccinated with Brucella melitensis Rev-1 vaccine

This study was conducted to investigate the effect of probiotic prepared from Lactobacillus acidophilus &amp; usedas immunostimulator to improve the humeral &amp;cellular immune response in lambs vaccinated with the Rev-1 vaccine.Lactobacillus isolated from intestinal contents of sheep after culturing on specific media &amp;study the morphological &amp;biochemical characteristic Lactobacillus acidophilus was selected as probiotic after definitive diagnosis and dependingon their higher adherence ability to epithelial cells of the intestine and have high tolerance to low pH and bile salts .thensensitivity test of the strain against antibiotics was done The experimental study included 15 lambs (age 5-8 months)were equally divided into three groups and treated as following.* First group was vaccinated with Rev -1 vaccine (1ml contain 2×109CFU- s/c as single dose ) and ioculated orally withprepared probiotic (2 ×109 CFU /lamb ) at one week before vaccination and continued daily to end of experiment(14weeks).*Second group was vaccinated only with Rev -1 vaccine at same way in first group. *Third group was inoculated with 1ml PBS S\c as control group. Clinical and immunological tests were conducted to all lambs at 0 times &amp; after 2, 6, 10, 14weeks. The results of clinical tests revealed that there is increase in body temperature, respiratory &amp;pulse rates ofvaccinated animals only but the second group show the highest parameters. The vaccinated lambs shows humeralimmune response by giving a positive results to rose Bengal test &amp; by using Passive haemagglutination test, the firstgroup revealed high significant antibody titer (716.80±125.413) than the second group and third group (p&lt;0.05 ) . andthe results of cellular immune response, as detected by delayed type hypersensitivity test which give a positive results invaccinated groups but the first group was significantly higher (p&lt;0.05 )at than rates in redness area and thicknesscompared with the second group E. rosette test showed significant increase at(p&lt;0.05 ) of activated lymphocyte infirst group ( 51.976±4.619 )compared with second (41.22±3.6865). Serum total protein concentration was increased infirst group (9.7358±0.2615) compared with other groups.