Serum Agglutination Titres Research Articles

Generation of Biotechnology-DerivedFlavobacterium columnareGhosts by PhiX174 GeneE-Mediated Inactivation and the Potential as Vaccine Candidates against Infection in Grass Carp

Flavobacterium columnare is a bacterial pathogen causing high mortality rates for many freshwater fish species. Fish vaccination with a safe and effective vaccine is a potential approach for prevention and control of fish disease. Here, in order to produce bacterial ghost vaccine, a specific Flavobacterium lysis plasmid pBV-E-cat was constructed by cloning PhiX174 lysis gene E and the cat gene with the promoter of F. columnare into the prokaryotic expression vector pBV220. The plasmid was successfully electroporated into the strain F. columnare G4cpN22 after curing of its endogenous plasmid. F. columnare G4cpN22 ghosts (FCGs) were generated for the first time by gene E-mediated lysis, and the vaccine potential of FCG was investigated in grass carp (Ctenopharyngodon idellus) by intraperitoneal route. Fish immunized with FCG showed significantly higher serum agglutination titers and bactericidal activity than fish immunized with FKC or PBS. Most importantly, after challenge with the parent strain G4, the relative percent survival (RPS) of fish in FCG group (70.9%) was significantly higher than FKC group (41.9%). These results showed that FCG could confer immune protection against F. columnare infection. As a nonliving whole cell envelope preparation, FCG may provide an ideal alternative to pathogen-based vaccines against columnaris in aquaculture.

Haemorrhagic septicaemia in the hybrid surubim (Pseudoplatystoma corruscans×Pseudoplatystoma fasciatum) caused by Aeromonas hydrophila

Intensive culture of the hybrid surubim (Pseudoplatystoma corruscans × Pseudoplatystoma fasciatum) in Brazil is responsible for the occurrence of diseases and consequent economic losses. However, the causative agents are not well known. The objective of this study was to isolate and to characterize the pathogenic agent responsible for mortalities in cultured surubim and to demonstrate its virulence. Ten fish from a fish farm located in the Mato Grosso do Sul State (Brazil) were collected and 14 haemolytic bacteria characterized as Aeromonas hydrophila were isolated from the kidneys (eight) and brain (six). As an experimental challenge, fish weighing 98.1±23.6 g were injected with 1 mL of saline solution and 2 × 102, 2 × 104, 2 × 106 and 2 × 108 CFU A. hydrophila mL−1. Fish infected with 2 × 108 CFU showed increased external and internal symptoms and mortality of 50±12.5% after 96 h. Increased A. hydrophila concentration was responsible for a decrease in haematocrit percentage and erythrocyte number, lymphocytes and eosinophils, as well as an increase in monocytes, neutrophils, serum agglutination titre and serum antimicrobial activity. It was concluded that A. hydrophila was responsible for characteristic symptoms of bacterial haemorrhagic septicaemia as well as important haematological and immunological alterations, which led to surubim mortality.

Unilateral Optic Neuritis as a Presentation of Neurobrucellosis

Neurobrucellosis manifesting as optic neuritis is a rare disease in childhood. We report a case of neurobrucellosis in a 11 year old girl leading to visual impairment and headache. Physical examination revealed mild oedema of right tibiotarsic joint and optic neuritis. Investigations showed CSF pleocytosis and a Brucella serum agglutination titer of 1/640. Complete reversal of the symptoms was observed after appropriate antibiotic treatment. To our knowledge only four cases of neurobrucellosis manifesting with visual impairment in childhood are previously reported in literature.

Simultaneous analysis of the nasal shedding kinetics of field and vaccine strains of Bordetella bronchiseptica

Groups of four two-week-old puppies were administered serial dilutions of an intranasal vaccine containing live Bordetella bronchiseptica and canine parainfluenza virus vaccine and housed individually in isolator cages. Three vaccinated...

Hematological and immunological responses of Nile tilapia after polyvalent vaccine administration by different routes

The efficacy of a polyvalent bacterin vaccine against Aeromonas hydrophila, Pseudomonas aeroginosa and Enterococcus durans administered by different routes in Nile tilapia was assessed by analyzing hematological and immunological parameters 7 and 21 days after vaccination. Treatments consisted of: non-vaccinated tilapia; tilapia vaccinated by intraperitoneal injection with 2x10(8) formalin-inactivated bacteria·mL-1; tilapia vaccinated orally with 2x10(7) formalin-inactivated bacteria·g-1, feed for 5 days; tilapia vaccinated by immersion bath in 2x10(7) formalin-inactivated bacteria·mL-1, for 20 minutes. Vaccinated fish groups presented higher hematocrit, number of erythrocytes and leukocytes than the non-vaccinated group. Serum agglutination titer of intraperitoneally vaccinated fish was higher on both evaluation periods for the three bacteria strains. Only on day 21 post-vaccination fish from the oral and immersion vaccination groups presented higher serum agglutination titer than the non-vaccinated fish for A. hidrophyla and E. durans. Serum antimicrobial activity in vaccinated fish was higher for P. aeroginosa and E. coli than in non-vaccinated fish on both evaluation periods. The different vaccine administration routes stimulated hematological and immunological responses in Nile tilapia 21 days post-vaccination, but intraperitoneal vaccination presented higher total number of leukocytes, lymphocytes and serum agglutination titer.

An Inactivation Method of Edwardsiella tarda Vaccine for Fish

Hossain, M.M.M., Kawai, K. and Oshima, S. 2009. An inactivation method of Edwardsiella tarda vaccine for fish. J. Appl. Anim. Res., 35: 137–142. The vaccine potential of inactivated E. tarda V-1 strain produced by pressure with French pressure cell (No 5501, OHTAKE Mfg, 941, Tokyo, Japan) was investigated. A major antigen at 37 kDa of E. tarda was obtained by SDS-PAGE and Western blot analysis. Pressure (PKC) killed E. tarda (600 kgf/cm2 for 5 min) showed enough antigen sustainability with moderate killing activity. Fish immunized with PKC showed no significant differences (P<0.05) with fish immunized with formalin (FKC) and low ampearage electric current (ECKC) in serum agglutination titers, but showed significantly higher bactericidal activity than fish immunized with FKC and ECKC. This study suggests pressure killed E. tarda (PKC) as a new vaccine candidate.

Brucellosis in pregnancy: a 6-year clinical analysis

To review our experience with brucellosis in pregnancy and to characterize the risk factors, clinical presentations, the rates of possible perinatal complications, and the effect of hospitalization on pregnancy outcomes. We reviewed the medical records of 21 pregnant women at a tertiary care hospital and 12 at a maternity hospital, who presented with acute, subacute, chronic, or relapsing brucellosis. Their risk factors and clinical presentations were defined. The reproductive outcomes of 29 cases were compared within themselves according to the hospitals they were managed and with the outcomes for all women followed in the maternity hospital for the period from January 2008 through December 2008. Consumption of unpasteurized dairy products had occurred in 92.3% of the cases. Spontaneous abortion, intrauterine fetal death, and preterm delivery rates were 24.14, 3.45, and 6.9%, respectively. Only spontaneous abortion rate substantially exceeded that among the general population of pregnant women in our maternity hospital (P<0.05). Hospitalization did not affect pregnancy outcomes significantly (P>0.05). Brucellosis in pregnancy is associated with increased incidence of spontaneous abortion without an association with the magnitude of serum agglutination titer, the clinical type of brucellosis and hospitalization. In endemic areas with inhabitants of low socioeconomic class and low educational level, educating women of childbearing age about brucellosis may help to prevent the disease and its complications in pregnancy.

Brucella Meningitis and Papilledema in a Child

To report a rare case of Brucella meningitis with papilledema in a child and to discuss the findings of neurobrucellosis in children. A 6-year-old girl was admitted with headache, fever, and vomiting for 1 week. Her family reported intake of raw unpasteurized goat's milk in the past. Meningeal signs were strongly positive. Bilateral moderate optic disc edema with flame-shaped hemorrhages was observed. The diagnosis of Brucella infection was established by positive blood culture for Brucella species, serum agglutination titer of antibodies to Brucella >1:160, and positive CSF culture. After treatment consisting of trimethoprim-sulfamethoxazole, rifampin, and doxycycline, the patient's condition gradually improved. One month later, the papilledema disappeared. One year after presentation, the patient remains free of symptoms. Brucella meningitis must be ruled out in symptomatic patients reporting ingestion of raw unpasteurized goat's milk. Papilledema is a frequent clinical feature, but irreversible visual impairment is extremely rare.

Protection of tilapia ( Oreochromis mosambicus) from edwardsiellosis by vaccination with Edwardsiella tarda ghosts

The vaccine potential of Edwardsiella tarda ghosts produced by gene E mediated lysis was investigated using tilapia ( Oreochromis mosambicus). Tilapia immunized with E. tarda ghosts (ETG) and formalin killed E. tarda (FKC) vaccines showed significantly higher serum agglutination titers than control fish. Fish immunized with ETG showed no significant differences with fish immunized with FKC in serum agglutination titers, but showed significantly higher bactericidal activity than fish immunized with FKC. Furthermore, fish immunized with ETG showed higher protection than fish immunized with FKC. As this promising type of a non-living whole cell envelope preparation seems to be favorable over conventional vaccines, we suggest E. tarda ghosts as a new vaccine candidate.

Genetical and functional investigation of fliC genes encoding flagellar serotype H4 in wildtype strains of Escherichia coli and in a laboratory E. coli K-12 strain expressing flagellar antigen type H48

BackgroundSerotyping of O-(lipopolysaccharide) and H-(flagellar) antigens is a wideley used method for identification of pathogenic strains and clones of Escherichia coli. At present, 176 O- and 53 H-antigens are described for E. coli which occur in different combinations in the strains. The flagellar antigen H4 is widely present in E. coli strains of different O-serotypes and pathotypes and we have investigated the genetic relationship between H4 encoding fliC genes by PCR, nucleotide sequencing and expression studies.ResultsThe complete nucleotide sequence of fliC genes present in E. coli reference strains U9-41 (O2:K1:H4) and P12b (O15:H17) was determined and both were found 99.3% (1043 of 1050 nucleotides) identical in their coding sequence. A PCR/RFLP protocol was developed for typing of fliC-H4 strains and 88 E. coli strains reacting with H4 antiserum were investigated. Nucleotide sequencing of complete fliC genes of six E. coli strains which were selected based on serum agglutination titers, fliC-PCR genotyping and reference data revealed 96.6 to 100% identity on the amino acid level. The functional expression of flagellin encoded by fliC-H4 from strain U9-41 and from our strain P12b which is an H4 expressing variant type was investigated in the E. coli K-12 strain JM109 which encodes flagellar type H48. The fliC recombinant plasmid carrying JM109 strains reacted with both H4 and H48 specific antisera whereas JM109 reacted only with the H48 antiserum. By immunoelectron microscopy, we could show that the flagella made by the fliC-H4 recombinant plasmid carrying strain are constituted of H48 and H4 flagellins which are co-assembled into functional flagella.ConclusionThe flagellar serotype H4 is encoded by closely related fliC genes present in serologically different types of E. coli strainswhich were isolated at different time periods and geographical locations. Our expression studies show for the first time, that flagellins of different molecular weigth are functionally expressed and coassembled in the same flagellar filament in E. coli.

Immune parameters in carpet shell clams naturally infected with Perkinsus atlanticus

Defence parameters of non-infected clams (Ruditapes decussatus) and clams heavily infected withPerkinsus atlanticus were assessed. Cellular (haemocyte density and phagocytic activity) and humoral (lysozyme and anti-bacterial activities, protein concentration and agglutination titre) parameters were measured in clams collected in an area enzootic for P. atlanticus. The infection intensity of each clam was assessed, and the immune parameters measured in the most infected clams were compared with those measured in the non-infected ones. Only the serum anti-bacterial activity and the agglutination titre were significantly different between infected and non-infected clams. The phagocytic rate, haemocyte density, lysozyme concentration and protein concentration were not statistically different but they showed the same trend in the two trials performed. Phagocytic rate, haemocyte concentration and anti-bacterial activity were higher in non-infected clams, while they had lower lysozyme concentration, serum protein concentration and agglutination titre than infected clams. Although infected and healthy clams were not different for every parameter measured, probably due to the high variability among individuals, P. atlanticus seems to affect the clam immune system, at least in advanced stages of the infection.

Experimental hepatitis induced by Campylobacter jejuni infection in Japanese quail (Coturnix coturnix japonica).

To establish an experimental model for vibrionic hepatitis caused by Campylobacter jejuni, Japanese quails (Coturnix coturnix japonica) were inoculated with C. jejuni strains isolated from chicken hepatitis (BL107) and human diarrhea (HP5113). Necrotic liver lesions were formed by intra-pancreaticoduodenal vein injection by which the bacteria reached the liver directly via the portal vein, but not by intra-gastric infection. These liver lesions were observed from day 1 to 7 after the infection. The pathological changes were weak and no clinical signs were observed throughout the experimental period. By immunohistochemistry, the bacterial antigens were detected in the hepatocytes, and intercellular spaces between the hepatocytes, and in the macrophages during the early stage of the infection. When focal hepatocyte necrosis was formed, the antigen was detected more frequently in the intact hepatocytes at the periphery of the lesions than within necrotic foci. The bacteria were not detected from the liver, spleen or blood according to raising the serum agglutination titer. In contrast, the bacteria immediately invaded the bile in 5 min post-infection and were retained in the gallbladder for long periods. The present study showed that necrotizing hepatitis was formed by intra-pancreaticoduodenal vein infection of the quail with C. jejuni.

&lt;I&gt;Bordetella bronchiseptica&lt;/I&gt;の莢膜抗原形成に及ぼすスルファモノメトキシンの影響

The authors reported that K antigen formation of sulfamonomethoxine (SMMX)-resistant Bordetella bronchiseptica is inhibited by SMMX in vitro. The present study was conducted to determine whether inhibition occurs also on the pig's concha nasalis in vivo, using experimentally infected pigs. SMMX in form of a feed added at the rate of 500 ppm was administered consecutively from one day before bacterial inoculation to the day of autopsy, which was performed 11 and 25 days after bacterial inoculation. The SMMX-administered group showed distinct inhibition of bone atrophy of the concha nasalis (AR lesion) both macroscopically and electron microscopically, in contrast to the control group. Decrease in viable cell count of B. bronchiseptica in the concha nasalis was noted in the former group. The serum agglutination titer in the former group decreased to a low value. K antigen formation of SMMX-resistant B. bronchispetica would thus appear to be inhibited in the presence of SMMX on the pig's concha nasalis, leading to inhibition of AR lesions.

Exacerbation ofMycoplasma gallisepticuminfection in Turkeys by rhinotracheitis virus

Groups of 1-day-old turkey poults from a parent flock free of antibodies to turkey rhinotracheitis virus (TRTV) and the pathogenic mycoplasmas, were infected by eyedrop with virulent TRTV, with Mycoplasma gallisepticum (Mg) or with both agents together. Dual infection resulted in increased morbidity compared with those groups given single infections. The presence of the Mg in the dual infection had no apparent effect on the pathogenesis of the virus, but the virus caused the Mycoplasma to be more invasive. Mg infection caused a transient depression in TRTV ELISA antibody titres at 29 days post-inoculation. At 14 days post-infection Mg haemagglutination inhibition (HI) and rapid serum agglutination (RSA) titres were higher (P <0.01) in the mixed infection group compared with those infected with Mg alone, but there was no significant difference between ELISA antibody titres of these two groups.

Use of an ELISA‐based assay for the detection of antibody‐secreting cells in channel catfish, Ictalurus punctatus (Rafinesque)

Abstract. An ELISA‐bascd plaque assay for the detection and quantification of antibody‐secreting cells has been adapted for use in fish. The assay involves incubating catfish lymphocytes in 24‐well plates previously coated with the antigen of interest. Cells producing antibody to the antigen leave an immunological fingerprint of bound antibody which is detected through the use of an ELISA technique to yield a colored plaque (Elisaplaque). Specificity of the assay was established by demonstrating that cells taken from fish vaccinated with an antigen exhibit the greatest response on plates coated with the given antigen. A strong positive correlation was also demonstrated between the number of Elisaplaques generated and serum agglutination titre. Using the ELISA plaque assay, it was found that antibody‐secreting lymphocytes located at a different density interface and behaved differently from other lymphocyte populations when separated through discontinuous Percoll gradient centrifugation. Among the haematopoietic organs examined, the head kidney appeared to produce more antibody‐secreting cells per million lymphocytes than did spleen or peripheral blood lymphocytes.

Effect of Anticapsular Antibodies on Neutrophil Phagocytosis of Staphylococcus aureus

One of the major virulence factors of Staphylococcus aureus is development of an exopolysaccharide capsule in vivo, which inhibits recognition of antibodies to highly antigenic cell wall by neutrophils. To circumvent this inhibition, an attempt was made to produce anticapsular antibodies. Three cows per group were immunized in midlactation by injections in the area of the supramammary lymph node and intramuscularly and were boosted on d 14, 42, and 70 with three variants of Smith S. aureus: compact, unencapsulated; diffuse, rigid capsule; and diffuse large clearing, exceptionally large flaccid capsule using dextran sulfate as adjuvant. Serum agglutination and ELISA titers of cows immunized with diffuse and diffuse large clearing increased after immunization and after each boost and remained elevated to the end of the experiment at 112 d. Phagocytosis of diffuse and diffuse large clearing, measured by flow cytometry, was enhanced by immunization with either organism. No antibody response to capsule or enhanced phagocytosis of diffuse developed in cows immunized with compact. However, anticompact antibodies were opsonic for diffuse large clearing. These data show that bovine antibodies to S. aureus capsule are opsonic for bovine neutrophils and that capsule plays a role in inhibition of cell-wall opsonization of S. aureus.

Development and Evaluation of a Monoclonal-Antibody-Based Enzyme-Linked Immunosorbent Assay for the Diagnosis ofRenibacterium salmoninarumInfection

A monoclonal-antibody-based enzyme-linked immunosorbent assay (ELISA) was developed for the diagnosis of bacterial kidney disease (BKD). This ELISA can detect Renibacterium salmoninarum antigen at concentrations as low as 0.05–0.1 μg/mL. During the 1988–1989 spawning season, 60 coho salmon Oncorhynchus kisutch, 60 chinook salmon O. tshawytscha, and 60 steelhead O. mykiss (Great Lakes rainbow trout) were caught and screened for BKD with the developed ELISA and a direct fluorescent antibody technique (FAT). Serum agglutination titers for R. salmoninarum were measured to determine any relationship between presence of antigen (R. salmoninarum) and humoral antibody to R. salmoninarum. Twelve of the coho salmon (20%), 48 of the chinook salmon (80%), and 7 of the steelhead (11.7%) were BKD-positive according to the ELISA. Only one steelhead (1.7%) was BKD-positive by FAT, whereas none of the coho salmon or chinook salmon were BKD-positive. It was concluded that the monoclonal-antibodybased ELISA was more sensitive than FAT. Antibody titers of these asymptomatic fish were variable. There was no correlation between antigen level and antibody titer.

Brucella melitensis Endogenous Endophthalmitis (With 1 color plate)

A 17-year-old female patient was examined and found to have Brucella-sp.-induced endophthalmitis in her left eye. The specific organism was Brucella melitensis, biotype 3, a known virulent organism. Serum agglutination titres from two independent laboratories were positive, yet of different levels: 1:320 and 1:1,280. Although brucellosis is thought to be eradicated in many countries, it prevails in agrarian environments, where it can be transmitted to man. This report is the first evidence to confirm that B. melitensis is a definitive causative organism of endogenous ocular infection.

Isolation of Brucella abortus from experimentally infected dromedary camels in Sudan: A preliminary report

Six camels were experimentally infected with two strains of Brucella abortus, four with S19 and two with a field bovine strain. In all cases antibody titres were detected within 6 to 11 days. Serum agglutination titres peaked between days 11 and 32 and complement fixation titres between days 11 and 52; both titres then declined steadily. No clinical signs were observed in the four camels inoculated with S19. Slight non-specific symptoms were seen in the two camels infected with the field bovine strain. On post mortem examination no gross lesions were observed although histopathological sections showed focal granulomata in the liver and a generalized lymphadenitis. The organism was recovered mainly from the lymph nodes of the head and genital tract.

The effect of sublethal endrin exposure on rainbow trout, Salmo gairdneri Richardson. II. The effect of altering serum cortisol concentrations on the immune response

The effect of altering serum cortisol concentrations on the immune response was elucidated in endrin‐ and non‐endrin‐exposed rainbow trout, Salmo gairdneri. Fish were immunized with 10 μg of Yersinia ruckeri O‐antigen following 30 days of treatment. The migration inhibition factor assay (MIF), plaque‐forming cell assay (PFC) and serum agglutination titres (SAG) were performed 2, 14 and 30 days post‐antigen inoculation. Endrin exposure was continued subsequent to antigen inoculation. Control fish were fed 20 and 35 mg kg−1 body weight day−1 of cortisol and metyrapone, respectively. Endrin‐exposed fish received 35 mg kg−1 body weight day−1 of metyrapone in their diet. Control fish receiving cortisol had significantly reduced MIF, PFC and SAG responses. The MIF response was completely restored in endrin‐exposed fish receiving dietary metyrapone. The PFC response and SAG titres were partially restored, 61 and 69% respectively, in endrin‐exposed fish receiving metyrapone. The results indicate that elevated serum cortisol concentration obtained in endrin‐exposed fish has a central role in repression of the immune response.