Effect of heat treatment on the surface antigens of Haemophilus pleuropneumoniae

Coagglutination and ring precipitation tests were used to study the effect of heat on the surface antigens of Haemophilus pleuropneumoniae strains employing the reference strains belonging to serotypes 1 to 7 and field isolates belonging to serotypes 1, 2, 3, 5, 6 and 7. By immunising rabbits with formalin-fixed whole-cell suspension, antibodies were obtained which sensitised Cowan I Staphylococcus aureus to coagglutinate antigen preparations which had not been heated, or heated at 56 degrees C, or boiled or autoclaved. Similar positive reactions were obtained with the ring precipitation test. Heating the cultures at 56 degrees C for one hour was best for exposing the most potent serotype-specific antigens in all the strains studied. All the reference strains and most of the field isolates possessed the thermostable type specific antigens which could withstand autoclaving for one hour. However, many field isolates belonging to serotype 1 did not possess this antigen. The apparent antigenic heterogeneity of serotype 1 strains based on the presence or absence of these thermostable antigens could be valuable in epidemiological investigations. It was shown that most potent serotype-specific antigens are present as freely diffusible material on the surface layer of the bacterial cells, which could easily be removed by washing in saline solution. Well washed bacterial cells devoid of surface materials are poor antigens. It is recommended that test strains should not be heated above 56 degrees C for serotyping because higher temperatures are liable to destroy the capsular antigen of some strains and render the culture untypeable.     

In Vitro Antimicrobial Activity of Sarafloxacin against Clinical Isolates of Bacteria Pathogenic to Fish

Abstract

An in vitro susceptibility assay of sarafloxacin (A-56620), a new 4-quinolone, was performed against five important bacterial species that are pathogenic to fish. A collection of 44 clinical isolates and five corresponding type strains were included in the study. The objectives were to determine the minimal inhibitory concentrations (MICs) of sarafloxacin by a drug microdilution method and to compare the MIC values at two different temperatures, 4 and 15°C. Sarafloxacin was active against all species tested and showed the following mean MIC values at 15 and 4°C, respectively, against the bacterial pathogens investigated: Aeromonas salmonicida subspecies salmonicida, 0.029 and 0.045 μg/mL; atypical A. salmonicida, 0.053 and 0.041 μg/mL; Vibrio anguillarum, 0.085 and 0.054 μg/mL; V. salmonicida, 0.125 and 0.123 μg/mL; and Yersinia ruckeri, 0.023 and 0.031 μg/mL. The MICs ranged from 0.0025 μg/mL (or less) for two strains of A. salmonicida salmonicida to 0.32 μg/mL for one strain of atypical A. salmonicida and one strain of V anguillarum. A decrease in antimicrobial activity was observed as the incubation temperature was lowered from 15 to 4°C; however, no significant statistical difference between the measured values was demonstrated.     

Serotypes of Campylobacter jejuni and C coli isolated from dogs

The serotypes of Campylobacter jejuni and C coli isolated from 56 dogs were established by the Penner serotyping scheme. A total of 37 C jejuni and 19 C coli were typed. Only two of the C coli strains were typable by the Penner method compared to 29 of 37 C jejuni strains. Pen 2 and 4 were the most predominant serotypes, constituting 41-4 per cent of the typable C jejuni strains. All but one of the C jejuni strains belonging to serotypes pen 1 and 2 were isolated from dogs with diarrhoea.     

Humoral Antibody Responses of Swine Infected Experimentally with Group E Streptococcus I. Whole Cell Agglutinin Response

Reference streptococcal antisera and sera collected from swine infected experimentally (by intranasal inoculation or contact exposure) with group E Streptococcus (GES) were studied in a tube agglutination system using whole GES cells.

Specificity studies revealed common group specific antigen among GES serotypes I and III, GES strains devoid of type specific antigen (untypable by ring precipitin testing) and group P and group U Streptococcus. The group specific antigens were not agglutinated by GES type specific antisera or by group specific antisera against Streptococcus groups A, B, C, D, F, G, H, K, L, M, N, or O. Results of the study suggested that GES serotypes I and III are invalid; i.e., they are devoid of type specific antigen.

Groug E Streptococcus type specific antigens II, IV, and V were agglutinated significantly only by their homologous antisera.

Experimentally infected swine developed significant titers against both the group and type specific antigen of GES. Antibodies appeared from three to eight weeks postexposure and persisted for the duration of the experiment (six months). The potential utilization of the whole cell agglutination (WCA) test for detection of GES carrier swine is discussed.

     

Actinobacillus (Haemophilus) pleuropneumoniae serotype-8 isolates and their antigenic relationships with other A pleuropneumoniae serotypes

Antigenic relationship of Actinobacillus (Haemophilus) pleuropneumoniae serotype-8 isolates with other serotypes was studied, using tube agglutination, with and without 2-mercaptoethanol, indirect hemagglutination with and without 2-mercaptoethanol, ring precipitation, coagglutination, and immunodiffusion tests. Serotype-8 isolates possessed serotype-specific, group-specific common antigens cross-reactive with serotypes 3 and 6 and species-specific common antigens cross-reactive with other serotypes. Absorption studies were done to study the antigenic relationship of serotype 8 with serotypes 3 and 6. Rabbit antisera against whole-cell (WC) suspensions of reference strains of serotypes 3, 6, and 8 were used for absorption studies with WC and boiled WC suspensions of homologous and heterologous serotypes. Unabsorbed and absorbed sera were tested for antibodies against WC and boiled WC antigen preparations of serotype 8, using various serotests. Absorption studies revealed that serotype-8 strains possessed 2 main types of epitopes, one of which was serotype-specific and did not have cross-reactivity with other serotypes. The second type of epitopes was group specific and was cross-reactive with serotypes 3 and 6.     

Role of Low-Molecular-Weight Ciliostatic Toxins in Vibriosis of Bivalve Mollusks

Abstract

Toxins play a major role in bacillary necrosis of hatchery-reared bivalve larvae. Two principal types of toxin of Vibrio spp. have been identified: A proteinase (molecular weight = 40,000) that degrades connective tissue and at least one ciliostatic toxin (molecular weight = 500–1,000). The ciliostatic toxin is stable at 100°C for 10 min and probably accounts for the cessation of feeding and swimming in the early stages of bacillary necrosis. Ciliostatic toxins were produced by 17 of 20 Vibrio spp. that are pathogenic to fish or that were isolated from diseased mollusks and included Vibrio tubiashii, V. anguillarum, and V. alginolyticus. Of 53 Vibrio and Aeromonas isolates not associated with bivalve infections, only 15 produced a ciliostatic toxin.     

Biochemical and serological properties of Actinobacillus pleuropneumoniae biotype 2 strains isolated from swine

The biochemical and serological properties of 21 strains of Actinobacillus pleuropneumoniae biotype 2 isolated from haemorrhagic necrotic pleuropneumonia of swine were examined. For serologic typing, the indirect haemagglutination (IHA) and the double gel-diffusion tests were used. On the basis of their soluble surface antigens, our A. pleuropneumoniae biotype 2 isolates could be assigned to two proposed serotypes. Serotype 1 comprised 11 strains and serotype 2 comprised 10 strains. All strains contained two surface antigen components. In the strains belonging to serotype 1, one of the antigens was identical with the serotype-specific antigen of Pasteurella haemolytica T4. Both antigens of serotype 2 strains proved to be type-specific. Four strains received from Switzerland, including the holotype strain of A. pleuropneumoniae biotype 2, and three strains isolated from swine in the G.D.R. belonged to serotype 2. Both the double gel diffusion and the IHA tests detected a 2-way cross-reaction between biotype 1, serotype 2 and biotype 2, serotype 2 strains of A. pleuropneumoniae, which could be eliminated using cross-absorbed sera.     

Non‐O157 Shiga Toxin‐producing Escherichia coli Isolated from Diarrhoeic Calves in Argentina

Faecal samples from 76 diarrhoeic calves belonging to 36 farms located in the Pampas plain, Argentina, were examined for Shiga toxin‐producing Escherichia coli (STEC). A total of 15 STEC strains were isolated from 12 (15.8%) calves which came from six different farms. All stx positive strains assayed by PCR were also positives in the Vero cell cytotoxicity test. The majority (60.0%) of the STEC strains carried the stx₁ gene. Twelve (80.0%) of the STEC isolates which belonged to serotypes O5:H‐ (n = 4), O26:H11 (n = 4), O26:H‐ (n = 1), O111:H‐ (n = 2), and O123:H38 (n = 1) were also enterohaemolysin (EHly) positive and carried the gene encoding for intimin (eae). All the stx positive strains were negative for the bfpA gene. Localized adherence to HEp‐2 cells were observed in 83.3% of the eae+ STEC strains. STEC belonging to serotype O5:H‐ showed atypical biochemical properties, including urease production. Urease was also produced by two strains belonging to serotypes O153:H? and non‐typeable, respectively. Resistance to three or more antibiotics was observed in 12 (80.0%) of the STEC isolates. Most of the serotypes of STEC recovered in this survey carried virulence traits that are associated with increased human and bovine pathogenicity. The present study shows that highly virulent STEC strains are being shed by diarrhoeic calves from farms located in a high incidence area of human STEC infections.     

Physiological and Immunological Effects of Adjuvanted Aeromonas salmonicida Vaccines on Juvenile Rainbow Trout

Abstract

The effects of injectable vaccines against Aeromonas salmonicida on oxygen consumption, growth, kidney lysozyme activity, and anti-A. salmonicida plasma antibody titers of juvenile rainbow trout Oncorhynchus mykiss were examined. The vaccines were A. salmonicida bacterin only, bacterin adjuvanted with levamisole, bacterin in emulsified oil, microencapsulated bacterin, microencapsulated bacterin with muramyl dipeptide, microencapsulated bacterin with β-1,3-glucan, and microencapsulated bacterin with Vibrio anguillarum lipopolysaccharide (LPS). The greatest and broadest ranges of responses were caused by the microencapsulated bacterin with V. anguillarum LPS. Oxygen consumption rates and specific growth rates were significantly higher over the course of 1 month among fish treated with the LPS vaccine. These fish also maintained a higher anti-A. salmonicida plasma antibody titer and kidney lysozyme activity for a substantially longer period than fish receiving the other treatments.     

Cell-Surface-Associated Properties of Fish Pathogenic Bacteria

Abstract

Pathogenicity assays showed that 33 of 42 potentially pathogenic strains of bacteria tested were virulent to rainbow trout Oncorhynchus mykiss. Regardless of their degree of virulence to fish, strains of motile Aeromonas, A. salmonicida, and Vibrio anguillarum were moderately hydrophobic. Only 46 and 25°10 of the strains were able to hemagglutinate human and trout erythrocytes, respectively. Hydrophobicity and hemagglutination were practically absent in isolates of Yersinia ruckeri. A notable number of the strains positively adhered to salmonid (51%) and nonsalmonid (55%) fish cells. Whereas the treatment of the bacteria with proteinase K or trypsin did not decrease the hydrophobicity of the isolates, within motile Aeromonas and A. salmonicida species, strains with both protease-sensitive and -resistant hemagglutinating and adhesive abilities occurred. The effects of heat and sugars on hemagglutinating and hydrophobic properties varied within all bacterial groups. Although treatment of strains with D-mannose or L-fucose had distinct effects on adhesiveness according to the bacterial species and the cell system used, none of the heat-treated (80°C for 15 min) bacteria lost their capacity to adhere to cultured fish cells. The results showed that there was no direct relationship between any of the cell surface properties analyzed and the degree of virulence of the strains.     

Growth Inhibition of Bacterial Fish Pathogens and Quorum-Sensing Blocking by Bacteria Recovered from Chilean Salmonid Farms

The main goal of this study was to find bacterial isolates with the ability to inhibit the growth of the fish pathogens Aeromonas hydrophila, Vibrio anguillarum, and Flavobacterium psychrophilum and to inhibit the blockage of the quorum-sensing (QS) system. A total of 80 gram-negative strains isolated from various freshwater Chilean salmonid farms were studied. We determined that 10 strains belonging to the genus Pseudomonas inhibited at least one of the assayed fish pathogens. Of these, nine strains were able to produce siderophores and two strains were able to inhibit the growth of all assayed pathogenic species. When the 80 strains were examined for QS-blocking activity, only the strains Pseudomonas sp. FF16 and Raoultella planticola R5B1 were identified as QS blockers. When the QS-blocker strains were analyzed for their ability to produce homoserine lactone (HSL) molecules, thin-layer chromatography analysis showed that both strains were able to produce C6-HSL– and C8-HSL–type molecules. Strain R5B1 did not show growth inhibition properties, but strain FF16 also led to inhibition of growth in A. hydrophila and F. psychrophilum as well as to siderophore production. Pseudomonas sp. FF16 exhibited potentially useful antagonistic properties and could be a probiotic candidate for the salmon farming industry.

Received July 31, 2014; accepted December 17, 2014     

Pathogenicity of Members of the Vibrionaceae Family to Cultured Juvenile Sablefish

Sablefish Anoplopoma fimbria are a prized seafood species due to their high oil content and white flaky flesh. Raising these species in culture can help to provide an important source of protein for humans and relief to declining wild fish populations. Understanding the environmental factors that influence the production of Sablefish is important for successful culturing. The significance of host–pathogen interactions in Sablefish culture and the resulting environmental implications are unknown. Pathogens could potentially cause losses of cultured Sablefish stocks due to disease, while Sablefish cultured in net pens may also serve as reservoirs for pathogens and potentially transmit disease to wild fish species. In this initial study, the susceptibility of juvenile Sablefish to three bacterial pathogens from the family Vibrionaceae was examined. Listonella anguillarum, Vibrio ordalii, and V. splendidus can pose serious economic threats to cultured fish and shellfish. Groups of juvenile Sablefish were exposed to five concentrations of each of the pathogens. Sablefish were susceptible to L. anguillarum, but were resistant to V. ordalii and V. splendidus at exposure concentrations of ≤1.32 × 10⁷ CFU/mL and ≤3.57 × 10⁶ CFU/mL, respectively. The greatest L. anguillarum concentration examined (8.7 × 10⁶ CFU/mL) resulted in 24% mortality in juvenile Sablefish. A 24% loss of Sablefish stock could significantly influence an aquaculture program. As determined by multiple logistic regression, the survival of Sablefish to L. anguillarum exposure was significantly affected by their body mass, and larger fish had a greater probability of survival. Aquaculture operations could employ various strategies to minimize the loss of juvenile Sablefish by accounting for their size and known susceptibilities to pathogens.

Received December 9, 2014; accepted February 7, 2015     

Serologic examination of the Westphal-type lipopolysaccharides of Pasteurella multocida.

The serologic specificities of the Westphal-type lipopolysaccharides from 16 serotypes of Pasteurella multocida were compared with those reported for the heat-stable typing antigens in the gel diffusion precipitin test. Like the heat-stable typing antigen, the Westphal-type lipopolysaccharide from each of the serotypes reacted only with its homologous antiserum in 14 of the 16 serotypes. The lipopolysaccharide from type strains representing serotypes 2 and 5 reacted with either of the corresponding typing sera, as did field isolates of these serotypes to a lesser extent. Although differences among the properties of these antigens were minor, the lipopolysaccharide appeared to be a major component of the heat-stable antigen responsible for the type specificity.     

Mode of Transmission of Vibriosis among Ayu Plecoglossus altivelis

Abstract

The mode of transmission of Vibrio anguillarum infection in ayu (Plecoglossus altivelis) and the portal of entry for the pathogen were investigated by the use of various challenge methods. The methods employed were immersion, direct contact, cohabitation, patch contact (filter paper soaked in bacterial suspension placed on part of a test fish for 1 min), and intubation. Based on the results of immersion, direct contact, and cohabitation challenges, it was concluded that water-borne infection is the primary mode of transmission of the disease. The patch contact challenge revealed that V. anguillarum penetrated the host through the skin, fins, anus, and gills. Among those, the skin and anus were determined to be the essential portals of entry for the pathogen. Artificial wounds of the skin surface caused by swabbing or scale removal facilitated invasion by the pathogen. Infection also was established by oral and anal intubations.     

辽西地区鸡大肠杆菌分离鉴定、药敏及IHA诊断液制备

本试验从辽西地区部分养鸡场采集具有典型临诊特征的鸡大肠杆菌病的病、死鸡病料,进行细菌分离培养和鉴定,分离到89株鸡大肠杆菌。通过血清型鉴定,鉴定出血清型的82株,血清型9种,分别为O₁、O₂、O₈、O₁₅、O₁₈、O₃₅、O₇₈、O₁₁₄、O₁₄₁,其中O₁₈、O₃₅型鸡大肠杆菌为辽西地区优势流行血清型。将分离纯化的O₃₅型鸡大肠杆菌超声裂解后,加入致敏双醛化处理过的红细胞,制备O₃₅型鸡大肠杆菌间接血凝诊断液,验证其特异性、敏感性和稳定性,完成诊断液的标准化。同时采用Kirby-Bauer纸片法,选择12种标准药敏片对此89株分离菌株进行药敏试验。结果表明,间接血凝诊断液特异性和敏感性好,质量稳定。而药敏试验结果显示,各地区均有耐药菌株的出现,仅对氟苯尼考、头孢噻呋、左旋氧氟沙星、盐酸沙拉沙星这4种药物敏感性较高。     

A National Surveillance of Shiga Toxin‐Producing Escherichia coli in Food‐Producing Animals in Japan

To assess the public health risk, the prevalence and anti‐microbial resistance of Shiga toxin‐producing Escherichia coli (STEC) among food‐producing animals were studied throughout Japan. Faecal samples were collected from healthy animals of 272 cattle, 179 pigs, and 158 broilers on 596 farms in all 47 Japanese prefectures. STEC were isolated from 62 (23%) cattle and 32 (14%) pig samples but from no chicken samples. Of the bovine isolates, 19 belonged to serotypes frequently implicated in human disease (O157:H7/non‐motile (NM)/H not typeable, O26:NM/H11/H21/H not typeable, O113:H21, and O145:NM). The eae genes were observed in 37% of bovine isolates; among them one O145:NM and all four O157 isolates possessed eae‐γ1, and one O145:NM, one O103:H11, and all five O26 isolates possessed eae‐β1 gene. Among the swine isolates, stx2e were dominant, and serotypes frequently implicated in human diseases or eae‐positive isolates were not observed. Bovine isolates showed less anti‐microbial resistance, but six isolates of 26:NM/H11 and O145:NM were multi‐resistant and may need careful monitoring. Swine isolates showed various resistance patterns; chloramphenicol resistance patterns were more common than in bovine isolates. This first national study of STEC in the Japanese veterinary field should aid our understanding of Japan's STEC status.     

Bovine adenoviruses—IV. Two mixed antigens for routine serodiagnosis by complement fixation reaction

The frequency of bovine adenovirus infections occurring in cattle has been grossly underestimated up to the present day. Primary isolation of serotypes belonging to bovine subgroup II is cumbersome. Hardly any laboratory has used all 8 officially-recognised bovine serotypes in seroneutralisation-tests, which should be done when this type-specific method is used for serodiagnosis. The complement fixation test, known as group-reactive from human adenovirus serology, has failed to disclose the true incidence of bovine infections, as until recently the importance of a novel additional group-reactive bovine adenovirus antigen had been undisclosed. Here we describe production, composition and performance of two mixed antigens for complement fixation reactions, which take into account recent findings by our team on peculiarities of bovine adenovirus antigens. Mixed antigen 1 contains bovine serotypes 1, 2 and 3 and detects group-specific antibodies of the classical mastadenoviruses. Mixed antigen 2 contains bovine serotypes 4, 5, 6, 7 and 8 and determines group-specific antibodies against the novel paramastadenoviruses. In addition, each antigen is capable of demonstrating complement-fixing type-specific antibodies in sera against the respective types included in each mixed antigen, with a net enhancing effect produced by mixing. Use of the 2 mixed antigens promptly shows whether bovine adenoviruses, presently recognised as well as unclassified, are involved in a given outbreak of respiratory or enteric disease of cattle. If needed, the responsible type may be determined in a second step of serological investigation.     

Diseases of Cultured Brown-Spotted Grouper Epinephelus tauvina and Silvery Black Porgy Acanthopagrus cuvieri in Kuwait

Abstract

Several diseases have been encountered in cultured brown-spotted grouper and silvery black porgy in the mariculture facilities of the Kuwait Institute for Scientific Research. More than 50% of juvenile brown-spotted grouper cultured in concrete tanks died during an initial outbreak of the protozoan parasite Cryptocaryon irritans. Formalin treatment (35–50 mg/L, 5 h/d, twice a week) was used to control and prevent the disease. Formalin-treated fish experienced several reinfestations by this parasite, but no further deaths occurred. Brown-spotted grouper also suffered from severe eye lesions, including exophthalmia and opaqueness of the cornea. Only 35% of these fish cultured in fiberglass tanks did not have idiopathic lesions. Silvery black porgy cultured in floating cages in the open sea did not show any lesions from July to November 1985 apart from eroded fins, which increased in frequency with increasing stocking densities. However, a disease occurred during the winter of 1986, when the water temperature averaged 14.8°C, and 65% mortality resulted. Vibrio anguillarum, V. ordain, V. carchariae, V. damsela, and three other Vibrio spp. were isolated from diseased silvery black porgy. Deaths associated with broken isthmuses occurred among silvery black porgy cultured in floating cages in August 1987. There was no further incidence of this condition after the addition of vitamin C (500 mg/kg) of feed to the diet.     

Serotypes of Escherichia coli strains isolated from piglets with diarrhea in swine industrial farms

Serotypes of E. coli strains isolated from piglets, which died with symptoms of diarrhea in 9 swine industrial farms, were determined. Large numbers of serotypes (from 16 to 27) in individual farms were detected. The sets of serotypes from 9 investigated farms differed among each other significantly, depending on the farm and time of examination. It was found that more than one serotype of E. coli may exist in the pig body and contribute to the development of disease. The predominant serotypes, i.e. those comprising more than 10% of serologically determined strains, were found to exist in 6 of the investigated farms and not in the remaining ones. Among the predominant serotypes, particularly important seem to be strains with K88 antigen. For prophylaxis of piglet colibacteriosis in industrial farms in Poland two vaccines for sows are recommended: one containing the K88 antigen only and the other the following serotypes: 0149:K91,K88; 020:K57; 020:K83; 0157:K88; 01:K1; 0136:K78; 024:K?; 078:K80 and 0118:K? Strains belonging to these serotypes were the most prevalent in our strain collection.     

Investigation into the pathogenesis of Atrophic Rhinitis in pigs

Summary

In two groups of swine herds, herds with and without clinical AR the presence of Atrophic Rhinitis (AR) correlated with the presence of toxinogenic Pasteurella multocida (PM) and not with the Bordetella bronchiseptica (BB) infection.

Six BB‐ and eighteen PM‐strains have been investigated for AR pathogenicity. Broth cultures were injected intradermally in guinea‐pigs (GPST) orintranasally in 3‐week‐old colostrum deprived specific pathogen free (SPF) piglets.

The average atrophy of the ventral conchae (A VC) correlated with the GPST in 4BB‐ and 7 PM‐strains. One BB‐ and 2 PM‐strains were qualified as doubtful, the others as non‐AR pathogenic. With AR pathogenic BB‐ and PM‐strains clinical AR could be induced in 3‐ and 6‐week‐old piglets. AVC lesions (gradation> 1) could be induced with BB in piglets of 6 and with pathogenic PM in 16‐week‐old piglets. Six of seven AR pathogenic PM‐strains resembled Carter‐type D and one resembled type A. No significance was found between AR pathogenicity and somatic serotypes.

Intranasal instillations of cell‐free broth culture filtrates of AR pathogenic PM‐strains also caused AR in piglets. These filtrates also caused lethality in piglets and in mice lethalitytest (MLT) and induced a positive GPST. After heating the pathogenic effects of the filtrates disappeared. The name AR toxin has been introduced for this thermolabile, haemorrhagic dermonecrotic (HDNT) fraction of the AR inducing filtrates. The severity of the AR lesions depended on the amount of the AR toxin intranasally instilled in pigs.

Cross protecting antibodies obtained in rabbits against the AR toxins of two PM strains could be demonstrated by a toxin neutralisation test in the MLT and the GPST.

Broth cultures were injected intradermally in guinea‐pigs (GPST) or intranasally in 3‐week‐old colostrum deprived specific pathogen free (SPF) piglets.