Robert Higgins

An update on Streptococcus suis identification.

Streptococcus suis is a worldwide cause of a variety sular type 2 isolate was recovered in pure culture from of porcine infections. It has been isolated from cases lungs and kidneys of a 41⁄2-month-old aborted bovine of meningitis, bronchopneumonia, arthritis, pericarfetus and its placenta. This suggests that S. suis may ditis, endocarditis, polyserositis, septicemia, rhinitis, be pathogenic for more than 1 animal species. and abortion.20,22,24 Different alpha-hemolytic strepIn Canada, it was reported that 94% of 4-8-weektococci were ascribed to Lancefield groups R, S, RS, old clinically healthy piglets harbored S. suis in their and T in 1963.5 Other investigators working with capnasal cavities, and 79% of these isolates did not belong sulated streptococci similar to de Moor’s groups S and to the 9 capsular types.3 Recently, we demonstrated R realized that the polysaccharides involved in serothat almost 90% of these untypeable isolates belong to typing originated from the capsular material rather than only 4 of the new capsular types (types 17, 18, 19, and from the cell wall. They considered these isolates as a 21, unpublished data). Other investigators have atnew species (Streptococcus suis) within the Lancefield tributed the development of rhinitis4,7,22 to untypeable group D, with 2 capsular types: 1 and 2, respectively. S. suis isolates. At this time, it is not possible to know The de Moor’s group RS was also added as S. suis type whether any of the capsular types of S. suis belong to 1⁄2.26 In 1983, 6 new capsular types of S. suis origithe normal flora of the nasal cavity or whether they nating from diseased pigs were described. 20 In 1987 represent real pathogens. Of the new capsular types, the species was officially recognized, l4 but the authors types 9 and 22 are more frequently found in diseased demonstrated by DNA hybridization that S. suis was pigs than in healthy ones. An outbreak of S. suis type not closely related to group D streptococci. 9 infection recently occurred in swine in Canada? Despite the presence of 9 capsular types of S. suis, Although 22 of 23 capsular types of S. suis are present untypeable isolates were still frequently reported. These in North America, our studies revealed that almost isolates were recovered from diseased4,10,12,13,22,24,25 and 30% of isolates recovered from diseased pigs are still clinically healthy pigs.2,3,21 More recently, 14 new capuntypeable (unpublished data). sular types have been described.9 Some of the reference As the number of capsular types increases, serotypstrains originated from diseased pigs, whereas others ing becomes more complicated, and it could soon bewere from the nasal cavities of clinically healthy pigs. come limited to reference laboratories. Even biochemOne strain was isolated from a diseased calf (capsular ical identification, when used alone, can be misleading. type 20) and another from a human case of meningitis Thus, veterinary diagnostic laboratories face a difficult (capsular type 14). Isolates belonging to capsular type situation. There is an urgent need for a standardization 14 have recently been recovered from diseased pigs in of both the biochemical identification of S. suis and our laboratory as well as in Denmark and Belgium (Dr. the techniques for capsular typing. The purpose of this J. Henrichsen, personal communication, 1989). This paper is to summarize the knowledge of biochemical confirms that types other than capsular type 2 could characterization and serotyping of S. suis and to suggest be involved in a zoonosis.9 In addition to the bovine means to facilitate the proper identification of this incapsular type 20, several other S. suis isolates have fectious agent of growing importance. been recovered from ruminants.9,12 Capsular type 16 Streptococcus suis is a gram-positive coccus that ocwas recently isolated in our laboratory from lungs of curs singly, frequently in pairs, or occasionally in short a calf suffering from bronchopneumonia, and a capchains. The organism grows well in aerobiosis, but growth is often enhanced by microaerophilic condiFrom the Research Group in Swine Infectious Diseases, Faculty of Veterinary Medicine, University of Montreal, Saint-Hyacinthe, tions. The majority of strains are alpha-hemolytic on

Isolation of Streptococcus suis from diseased pigs in Canada.

A total of 260 isolates of streptococci collected over a 9-year period from diseased pigs submitted for necropsy were studied. Seventy-seven percent of isolates were identified as S. suis and 32% of S. suis isolates were retrieved in pure culture. S. suis was found more frequently in lungs and was often isolated in conjunction with Actinobacillus pleuropneumoniae, Pasteurella multocida, Escherichia coli and other microorganisms. A total of 151 (76%) of S. suis isolates could be serotyped within the 9 recognized serotypes. Serotype 2 was the most prevalent with 33%, followed by serotypes 3, 5 and 7. All isolates were sensitive to ampicillin, penicillin, cephradine, chloramphenicol and trimethoprim-sulfamethoxazole. Resistance to streptomycin, neomycin and tetracycline appeared to be very high.

Serological characterization of Actinobacillus pleuropneumoniae strains isolated from pigs in Quebec

A total of 3306 isolates of A. pleuropneumoniae originating from lung tissues of pigs that died of acute pleuropneumonia and 140 isolates recovered from tonsils or nasal cavities of apparently healthy pigs from chronically infected herds were serotyped. Various serotyping methods, such as slide agglutination, tube agglutination, ring precipitation, coagglutination, immunodiffusion, indirect hemagglutination and counterimmunoelectrophoresis either alone or in combination were used. The techniques used for serotyping continued to evolve during the last 10 years depending on the problem encountered in serotyping. Antisera prepared in rabbits against formalinized whole cell suspensions of reference strains of A. pleuropneumoniae of serotypes 1 to 12 were employed for serotyping. Serotype 1 was predominant ranging from 55 to 87% from year to year during the last 10 years with an average prevalence of 68%. Serotype 5 was second in prevalence ranging from 9 to 30% with a mean of 23%. Both subtypes of serotype 5 (5a and 5b) were present in Quebec. Serotypes 3, 6, 7, 8, 10 and 12 were isolated in small numbers together accounting for about 9%. Serotypes 4, 9 and 11 were not present. Cross-reactions were observed among isolates of serotypes 3, 6 and 8, and 1, 9 and 11 and were easily differentiated from each other by quantitation of type and group specific antigens by coagglutination and immunodiffusion tests. Serotypes 1, 5 and 7 were isolated most frequently from tonsils of pigs from chronically infected herds. Prevalence of different serotypes in different countries has also been reviewed.

Risk factors and impacts of clinical and subclinical mastitis in commercial meat-producing sheep flocks in Quebec, Canada.

We conducted a prospective observational study on clinical and subclinical mastitis in 30 commercial meat-producing sheep flocks from 2 regions of the province of Quebec, Canada. A total of 2,792 ewes selected in late gestation were followed from lambing to weaning of lambs. The incidence of clinical mastitis for the total lactation period (average of 58 days) ranged among flocks from 0 to 6.6%, with a median of 1.2%. The most frequently isolated bacteria from the cases of clinical mastitis, in pure or mixed culture, were Mannheimia haemolytica (26%), Staphylococcus aureus (23%), and coagulase-negative staphylococci (17%). Incidence of clinical mastitis was higher in ewes that gave birth to 3 or more lambs and from the Estrie region, and was associated with an increase in ewe mortality, an increase in lamb mortality at the litter level, and a decrease in lambs weaning weight for lambs born in multiple litter size or from ewes >or=4 years old. Among 354 selected ewes with clinically normal udder at the end of lactation, 28.8% had potentially pathogenic bacteria isolated from milk. The most prevalent bacteria were S. aureus (9.3%) and coagulase-negative staphylococci (9.3%). The risk of having a positive culture in at least one half was different between the two regions. Prevalence of ewes (n=261) with California Mastitis Test (CMT) positive result in at least one half was 24.1 and 14.9% using a cut-off of >or=1+ and >or=2+, respectively. Prevalence of culture-positive udder halves was 11.7% for CMT-negative compared with 53.6% for CMT 3+ halves. CMT status was positively associated with the isolation of coagulase-negative staphylococci, M. haemolytica, S. aureus, and various Streptococcus species, but not with other isolated bacteria. Additionally, prevalence of CMT-positive halves was higher in ewes from the Estrie region, aged of >or=4 years versus 1 year, having clinical mastitis previously detected in the lactation and/or with low body condition score. Lamb weaning weight was associated with CMT status of ewes, while weaning weight was not associated with milk culture results. More research is needed to understand the dynamic of milk SCC and IMI in ewes from meat-producing flocks, its economical impact and best ways to control it.

Production and characterization of two Streptococcus suis capsular type 2 mutants

Two avirulent mutants of Streptococcus suis capsular type 2 (M2 and M42) were produced from a highly virulent strain. Mutant M2, obtained after serial subcultures of the parent strain in the presence of rabbit anti-capsular type 2 serum, no longer possessed the type-specific capsular antigen, as demonstrated by serotyping methods and immunoelectron microscopy. The Lancefield group D antigen could not be detected on the cell surface of this mutant using the immunogold labelling technique. SDS-PAGE of lysozyme treated cells demonstrated that a 44 kDa protein which was present in the parent strain, was absent in mutant M2. Immunoblotting using rabbit whole cell homologous anti-serum revealed that the protein was strongly immunogenic. Mutant M2 was totally avirulent in mice, and the homologous antiserum completely failed to protect mice against challenge with the parent strain. However, mutant M42, obtained after passages of the parent strain at 42 degrees C, remained capsulated but lacked the same 44 kDa protein as mutant M2. The quantity of sialic acid present in the capsule was similar to that of the parent strain. Despite the presence of antibodies against the capsule, antiserum prepared against M42 only partially protected mice against a challenge with the parent strain. The 44 kDa cell wall protein could act as a virulence factor as well as an important immunogen of S. suis capsular type 2.

Virulence factors in Escherichia coli isolated from the blood of bacteremic neonatal calves

Twenty-five Escherichia coli isolates from the blood of critically ill bacteremic calves sampled in two separate studies on a calf-rearing farm housing over 15,000 calves, in the San Joaquin Valley, California were studied. Isolates were characterized for O serogroups and for pathotypes as determined by the presence of specific virulence factors including heat-labile enterotoxin (LT), heat-stable enterotoxins a and b (STa, STb), verotoxins 1 and 2 (VT1, VT2), cytotoxic necrotizing factor (CNF), aerobactin, intimin Eae and P, F17 and CS31A fimbrial adhesins, and resistance to bactericidal effects of serum. These isolates constituted a heterogeneous group. However, isolates were mostly aerobactin positive and often resistant to the bactericidal effects of serum. Isolates of pathotypes O78 (n=6), O119:CS31a (n=3), and P positive but O non-typeable (n=3) were associated with a high mortality rate. The remaining isolates belonged to diverse pathotypes, often possessing the adhesins P, F17, CS31A and Eae but belonging to O serogroups other than O78 and O119, and were less frequently associated with mortality. Although no virulence factor common to all isolates was identified, the capacity to use iron by the presence of aerobactin which is important to the capture of iron was a predominant factor. Moreover, certain pathotypes appear to be associated with primary colisepticemia whereas other pathotypes may cause a bacteremia without necessarily leading to septicemia.

Infections of Edwardsiella tarda among Brook Trout in Quebec

Acute bacterial septicemia is commonly diagnosed in brook trout Salvelinus fontinalis of Quebec, Canada. The agents most commonly isolated include Aeromonas salmonicida (furunculosis), Aeromonas hydrophila (motile aeromonad septicemia), and Pseudomonas species. Septicemia in brook trout caused by the gram-negative bacterium Edwardsiella tarda was diagnosed for the first time in the province of Quebec from two different fish farms producing stock for fee fishing establishments. Affected fish displayed nonspecific lesions associated with bacterial septicemia including hemorrhages on the gills and viscera and exophthalmia. Stress-associated immunosuppression due to an increase in summer water temperatures and lack of precipitation were considered as primary causes of these disease outbreaks.

Antimicrobial susceptibility of Streptococcus suis isolates

The antimicrobial activities of penicillin (PEN), ampicillin (AMP), cephalothin (CT), trimethoprim-sulphamethoxazole (TMP-SMX), streptomycin (STM), and gentamicin (GM) against 122 representative strains of Streptococcus suis, were compared by the agar dilution procedure. The current US National Committee for Clinical Laboratory Standards (NCCLS) breakpoints for non-enterococcal streptococci were used for PEN, AMP, CT, and TMP-SMX. Overall, 50% of strains were not fully susceptible to PEN, whereas these percentages for AMP and CT were 9% and 6% respectively. One strain was resistant to TMP-SMX. High-level GM resistance could not be detected, but more than 46% of strains were highly resistant to STM (MIC > 2000 mg l-1). This high percentage of resistance to STM precludes the use of this aminoglycoside-penicillin combination as empiric therapy in severe S. suis infections. These results should prompt microbiology laboratories to carry out antimicrobial susceptibility tests on a routine basis on S. suis isolates.

Proposal of Bisgaardia hudsonensis gen. nov., sp. nov. and an additional genomospecies, isolated from seals, as new members of the family Pasteurellaceae

Phenotypic and phylogenetic studies were performed on eight Gram-negative-staining, rod-shaped bacteria isolated from seals. Biochemical and physiological studies showed identical profiles for all of the isolates and indicated that they were related to the family Pasteurellaceae. 16S rRNA gene sequencing demonstrated that the organism represented a distinct cluster with two sublines within the family Pasteurellaceae with <96% sequence similarity to any recognized species. Multilocus sequence analysis (MLSA) including rpoB, infB and recN genes further confirmed these findings with the eight isolates forming a genus-like cluster with two branches. Genome relatedness as deduced from recN gene sequences suggested that the isolates represented a new genus with two species. On the basis of the results of the phylogenetic analysis and phenotypic criteria, it is proposed that these bacteria from seals are classified as Bisgaardia hudsonensis gen. nov., sp. nov. (the type species) and Bisgaardia genomospecies 1. The G+C content of the DNA was 39.5 mol%. The type strain of Bisgaardia hudsonensis gen. nov., sp. nov. is M327/99/2(T) (=CCUG 43067(T)=NCTC 13475(T)=98-D-690B(T)) and the reference strain of Bisgaardia genomospecies 1 is M1765/96/5 (=CCUG 59551=NCTC 13474).

Simultaneous flow cytometric measurement of Streptococcus suis phagocytosis by polymorphonuclear and mononuclear blood leukocytes

A simple flow cytometric method was used to study simultaneously the phagocytosis of Streptococcus suis serotype 2 by polymorphonuclear and mononuclear blood leukocytes from swine and humans. Using this method with a bacteria-to-leukocytes ratio of 10:1 and after 60 min of incubation, 80.2 +/- 2.8% of swine granulocytes and 77.0 +/- 2.8% of swine monocytes were shown to contain FITC-labelled S. suis serotype 2 strain 735. Using the same strain, FITC-labelled bacteria were found in 95.5 +/- 3.2% of human granulocytes and in 92.8 +/- 3.6% of human monocytes. The phagocytosis rates of avirulent and virulent strains of S. suis were not significantly different.