J. Goyache

Analysis of Genetic Diversity of Streptococcus suis Clinical Isolates from Pigs in Spain by Pulsed-Field Gel Electrophoresis

ABSTRACT Pulsed-field gel electrophoresis (PFGE) was used to investigate the diversity of Streptococcus suis isolates of various serotypes recovered from swine clinical samples in Spain. Capsular types 9 (64.9%) and 2 (14.8%) were the most frequently isolated serotypes followed by serotype 7 (5.9%) and serotype 8 (4.3%). The PFGE results of this study with 60 different pulsotypes indicate a great genetic diversity among the S. suis isolates, which is consistent with the broad distribution of S. suis in the swine population. Forty-five percent of the pulsotypes corresponded to single isolates, no pulsotype was common to all farms, and at least 3 different pulsotypes were isolated in 56% of herds in which more than 3 clinical isolates were analyzed. These results reveal a great diversity both between and within herds throughout the strains of S. suis studied, demonstrating that different strains of S. suis are associated with infection in pigs. Some pulsotypes were more frequently isolated and exhibited a wider distribution over herds than others, and were the unique or predominant strains in several herds, suggesting the existence of a prevalent or a few prevalent clones responsible for a large proportion of clinical cases. Overall, the great genetic heterogeneity of the clinical strains of S. suis, the isolation of different strains within the same herd, and the predominance of particular strains in some herds are evidence that infection by S. suis is a dynamic process and reinforce the idea that the epidemiology of S. suis infection is very complex.

Management of an outbreak of brucellosis due to B. melitensis in dairy cattle in Spain

Brucella melitensis is a major human and animal pathogen, with a wide host range that includes all domestic ruminant species, although small ruminants are its preferred hosts. Outbreaks in cattle due to B. melitensis have become a worldwide emerging problem particularly difficult to control due to the lack of knowledge on the epidemiology in this host species and of an effective vaccine. However, combination of molecular tools and strict biosecurity measures can help to solve these difficulties and eventually eradicate the disease from infected herds. In the present report, management of an outbreak in Spain involving four farms, more than 2000 cattle and several human cases is described. Application of Multiple Locus VNTR Analysis (MLVA) allowed identifying the most likely source of infection. Stamping out and test-and-slaughter strategies were applied, proving their usefulness to control the outbreak depending on infection level, and without the need of other alternative measures.

Detection of anti-Leishmania infantum antibodies in sylvatic lagomorphs from an epidemic area of Madrid using the indirect immunofluorescence antibody test

An outbreak of human leishmaniasis was confirmed in the southwest of the province of Madrid, Spain, between July 2009 and December 2012. Incidence of Leishmania infection in dogs was unchanged in this period, prompting a search for alternative sylvatic infection reservoirs. We evaluated exposure to Leishmania in serum samples from animals in the area with an indirect immunofluorescence test (IFAT). Using promastigotes from six culture passages and a 1/25 threshold titer, we found anti-Leishmania infantum seroreactivity in 9.3% of cats (4 of 43), 45.7% of rabbits (16/35) and 74.1% of hares (63/85). Use of promastigotes from >10 in vitro passages resulted in a notably IFAT lower titer, suggesting antigenic changes during extended culture. Postmortem inspection of seropositive animals showed no clinical signs of infection. The results clearly suggest that asymptomatic hares were the main reservoir in the outbreak, and corroborate IFAT as a sensitive serological surveillance method to detect such cryptic Leishmania infections.

In vitro infection of cells of the monocytic/macrophage lineage with bovine leukaemia virus.

The oncogenic retrovirus bovine leukaemia virus (BLV) primarily infects B cells. Most infected animals remain asymptomatic for long periods of time before an increase in circulating B cells or localized tumours can be observed. This long clinical latency period may be explained by cells of the monocyte/macrophage lineage (M/M) becoming infected and acting as a reservoir for the virus, as shown for other retroviruses (human immunodeficiency virus-1, feline immunodeficiency virus). M/M cells in different stages of differentiation (HL-60, THP-1, U-937, J774, BGM, PM2, primary macrophages of sheep and cows) were cultured with BLV produced by permanently infected donor cells (FLKBLV and BLV-bat(2)). Donor cells were inhibited from multiplying by either irradiation or treatment with mitomycin C. In other experiments, supernatant from donor cells containing virus was used. In co-culture with the donor cells, the less differentiated monocytic cells showed severe cellular changes such as differentiation, vacuolization, cell lysis and membrane blebbing; apoptosis was a frequent phenomenon. Budding and extracellular viruses were also observed. The more differentiated macrophage cells, although they showed less signs of infection by microscopy, had a complete BLV protein profile, as seen by Western blotting; bands corresponding to p24CA (Gag) and its precursors were clearly seen. In addition, gp51SU was identified by syncytia formation assays. It is concluded that M/M cells may be infected by BLV, the consequences of the infection differing according to the type of cell.

Weissella confusa Infection in Primate (Cercopithecus mona)

We describe systemic infection by Weissella confusa in a mona monkey (Cercopithecus mona) on the basis of microbiologic, molecular genetic, and histologic data. The same strain of W. confusa, as determined by pulsed-field gel electrophoresis, was isolated in pure culture from the primate’s brain, liver, spleen, and intestine. Histologic lesions showed inflammatory infiltrates mainly composed of neutrophils, indicating an acute septicemic process.

Characterization of Aerococcus viridans Isolates from Swine Clinical Specimens

ABSTRACT We present here the biochemical and genetic characterization and antimicrobial susceptibility of 58 isolates of Aerococcus viridans isolated in pure culture from different clinical specimens of normally sterile body sites of pigs. A. viridans isolates were commonly susceptible to β-lactam antimicrobials and exhibited a great genetic heterogeneity as determined by pulsed-field gel electrophoresis typing. The results indicate that A. viridans might be included in the list of possible etiological agents causing disease in pigs.

Assessment of genetic diversity of zoonotic Brucella spp. Recovered from livestock in Egypt using multiple locus VNTR analysis

Brucellosis is endemic in most parts of Egypt, where it is caused mainly by Brucella melitensis biovar 3, and affects cattle and small ruminants in spite of ongoing efforts devoted to its control. Knowledge of the predominant Brucella species/strains circulating in a region is a prerequisite of a brucellosis control strategy. For this reason a study aiming at the evaluation of the phenotypic and genetic heterogeneity of a panel of 17 Brucella spp. isolates recovered from domestic ruminants (cattle, buffalo, sheep, and goat) from four governorates during a period of five years (2002–2007) was carried out using microbiological tests and molecular biology techniques (PCR, MLVA-15, and sequencing). Thirteen strains were identified as B. melitensis biovar 3 while all phenotypic and genetic techniques classified the remaining isolates as B. abortus (n = 2) and B. suis biovar 1 (n = 2). MLVA-15 yielded a high discriminatory power (h = 0.801), indicating a high genetic diversity among the B. melitensis strains circulating among domestic ruminants in Egypt. This is the first report of the isolation of B. suis from cattle in Egypt which, coupled with the finding of B. abortus, suggests a potential role of livestock as reservoirs of several zoonotic Brucella species in the region.

Associations between biovar and virulence factor genes in Pasteurella multocida isolates from pigs in Spain.

Two hundred and five isolates of Pasteurella multocida from pigs were phenotypically and genetically characterised by determining their biovar, capsular type, virulence-associated genes and pulsed-field gel electrophoresis (PFGE) profiles. All isolates were identified as P multocida subspecies multocida and most were assigned to biovar 3 (58 per cent) and biovar 2 (39.5 per cent). Biovar 1 represented 2.4 per cent of the isolates. According to the capsular type, the great majority of the isolates (79.0 per cent) belonged to capsular type A, 18.5 per cent belonged to capsular type D and 2.4 per cent were of capsular type F. All isolates harboured ompH, psl, oma87, ptfA, nanB, nanH, tonB, hgbA, sodA and sodC genes, while none of them possessed the transferrin-binding protein gene tbpA. The prevalence of toxA, pfhaA and hgbB genes was variable (7.8, 40.5 and 60.5 per cent of the isolates, respectively). After PFGE typing, isolates of biovar 2 and 3 were grouped in two different clusters (A and B) at a level of 45 per cent similarity. In addition, isolates of biovar 2 and 3 exhibited statistically significant differences (P<0.05) in the virulence-associated hgbB and pfhA genes (biovar 3 was hgbB+ pfhA–, while biovar 2 was hgbB– pfhA+).

Distribution of serotypes of Streptococcus suis isolated from diseased pigs in Spain

C. Tarradas, DVM, A. Perea, DVM, C. Borge, PhD, B. Huerta, DVM, I. Luque, DVM, Departamento de Sanidad Animal, Facultad de Veterinaria, Campus Universitario de Rabanales, 14071 C6rdoba, Spain A. I. Vela, DVM, J. Goyache, DVM, L. Dominguez, DVM, J. F. FernAndezGaraizabal, DVM, Departamento de Patologia Animal I (Sanidad Animal), Facultad de Veterinaria, Universidad Complutense, 28040 Madrid, Spain Streptococcus suis is well known as a major cause of meningitis, septicaemia, endocarditis, reproductive disorders, pneumonia and arthritis in pigs (Higgins and Gottschalk 1999). It is also considered an important zoonotic agent (Arends and Zanen 1988), able to induce meningitis and septicaemia in human beings, and mainly affects pig handlers (Tarradas and others 2001). At present, 35 different serotypes, based on capsular antigens, are recognised (Higgins and others 1995); serotype 2 is the most frequently isolated capsular type in Italy, France and Denmark, while serotype 9 is more frequently isolated in Germany, the Netherlands and Belgium (Gogolewski and others 1990, Aarestrup and others 1998, Sihvonen and others 1998, Berthelot-Herault and others 2000, Wisselink and others 2000, Allgaier and others 2001). Previous studies carried out in Spain have demonstrated that serotype 2 is the most prevalent serotype associated with different disease conditions in pigs (Prieto and others 1993, Luque and others 1998). This short communication reports on 383 strains of S suis isolated from diseased pigs on 99 farms in Spain between 1998 and 2002 during the course of routine diagnostic procedures. All isolates were grown overnight on Columbia agar (Oxoid) and incubated with nalidixc acid, colistin sulphate (Oxoid) and 5 per cent defibrinated sterile sheep blood at 37°C in aerobic conditions for 18 to 24 hours. Biochemical identification of S suis was performed by conventional tests (Devriese and others 1991, Tarradas and others 1994) and by using the commercial identification system Rapid ID32 Strept (bioMerieux), according to the manufacturers instructions. Capsular typing was carried out by slide agglutination with specific rabbit antiserum against the reference strains of serotypes 1 to 34. Statistical analysis was performed to determine the relationship between S suis capsular types and clinical sources. The chi-squared test was used with sPss 11.0 software. Differences were considered significant when P<0 05. All but four of the 383 S suis isolates (98-9 per cent) could be serotyped using the 34 capsular types, and showed a great serotype diversity, with 15 different capsular types being identified (Table 1). Capsular types 9 (54 per cent) and 2 (19.3 per cent) were the most frequently isolated serotypes, followed by serotypes 7 (6.3 per cent), 8 (5.7 per cent), 3 (4.2 per cent) and 1/14 (3.6 per cent). The other serotypes were isolated at a frequency of 1 per cent or less (Table 1). Serotypes 19, 21 and 23 had not been isolated previously from diseased pigs in Spain. The prevalence of serotype 2 was approximately 50 per cent lower than that usually reported for this capsular type in Spain, while serotype 9 was isolated in a proportion much higher than that previously found (Prieto and others 1993, Luque and others 1998, Tarradas and others 2001). It is known that healthy carrier pigs harbouring the organism in their palatine tonsils are involved in the spread of S suis infection (Clifton-Hadley 1985, Gottschalk and Segura 2000, Berthelot-Herault and others 2001). The high frequency of serotype 9 observed in this study could be related to the increased importation of animals from countries in which this capsular type has a high prevalence, such as Germany, the Netherlands and Belgium (Aarestrup and others 1998, Wisselink and others 2000, Allgaier and others 2001). Recent studies based on the multilocus sequence typing scheme of S suis demonstrated genetic diversity among isolates, suggesting that capsular genes may move horizontally through the S suis population (King and others 2002), which would also explain the changes in the distribution of the serotypes. The isolates were recovered from pigs exhibiting a variety of clinical disease conditions (Table 1). Nervous disorders (52.2 per cent) and septicaemia (32.9 per cent) represented the majority of the cases, while other conditions associated with S suis infection included arthritis (2.9 per cent) and metritis ( 16 per cent). These results are similar to those of previous studies in Spain (Luque and others 1998). Of particular interest is the high frequency (10-4 per cent) of cases of endocarditis, characterised by the production of a valvular endocarditis, which had not been previously described, probably because endocarditis does not usually produce clinical signs in live animals (Pedersen and others 1981, Sanford and Park 1987). The relationships between serotypes and clinical disease conditions are shown in Table 1. A high number of isolates belonging to serotype 9 (68 per cent) were recovered from pigs with nervous disorders, while the septicaemia cases associated with this serotype represented a lower prevalence (19.8 per cent). Serotype 2 was also recovered from pigs with nervous disorders (32.4 per cent) and septicaemia (45.9 per cent). Endocarditis cases were associated with serotypes 2, 3, 7, 8 and 9. The statistical association between serotypes and clinical conditions was only determined for the most frequent serotypes. The majority of the strains of serotype 9 (68.1 per cent) were isolated from pigs with nervous disorders, while 63 6 per cent of the strains of serotype 8 were isolated from pigs with septicaemia. The association between these serotypes and clinical source was statistically significant (P<0.05). There was no significant association between strains of serotype 2 and 7 and the clinical source. In conclusion, this study shows that a varied distribution of S suis serotypes has caused a variety of infections in pigs over the past few years in Spain. Serotype 9 has emerged as the predominant serotype and new serotypes ( 19, 21 and 23) have been isolated from diseased pigs in the country. This pathogen remains the main cause of nervous disorders and

Growth of Staphylococcus aureus and Enterotoxin Production in Homemade Mayonnaise Prepared with Different pH Values

The ability of Staphylococcus aureus to grow and produce enterotoxins in homemade mayonnaise prepared at different pH values was studied. Ten enterotoxigenic strains, producing one or two enterotoxin types (A, B, C, or D) were inoculated into mayonnaise samples with pH adjusted to values ranging between 4.0 and 5.8, and incubated at 37°C for 7 d. Counts were made on days 1, 3, 5, and 7 and extracts were prepared on day 7 to detect enterotoxin by ELISA. An important difference was seen between those samples prepared with pH below or equal to 4.9 and those over or equal to 5.0; in the range of pH between 4.0 and 4.9 the average of staphylococcal population was 100 CFU/g; at pH 5.0 it was 1.6 × 105, and at pH 5.15 and above it was at least 8 × 106 CFU/g. Enterotoxin was detected only at initial pH over 5.15 and when final pH was not less than 4.7. The highest amount of enterotoxin corresponded to 157.8 ng of SEB/100 g of mayonnaise.