Polymerase chain reaction amplification of repetitive intergenic consensus and repetitive extragenic palindromic sequences for molecular typing of Pseudomonas anguilliseptica and Aeromonas salmonicida

The aim of this study was to evaluate the use of two molecular techniques, repetitive extragenic palindromic polymerase chain reaction (REP-PCR) and repetitive intergenic consensus PCR (ERIC-PCR), as epidemiological tools with which to discriminate among genetically distinct strains within two bacterial fish pathogens, Pseudomonas anguilliseptica and Aeromonas salmonicida. A total of 30 A. salmonicida and 52 P. anguilliseptica were analyzed. For P. anguilliseptica, three different major fingerprints were obtained with both techniques, which defined three genomic groups: one was composed of strains isolated from eels Anguilla spp., the second of strains from turbot Scophthalmus maximus and blackspot seabream (also known as red seabream) Pagellus bogaraveo, and the third of strains from other fish species, such as gilthead seabream (also known as gilthead bream) Sparus auratus, sea bass Dicentrarchus labrax (also known as European bass Morone labrax), and salmonids. In the case ofA. salmonicida, promising results were obtained with both techniques for subspecies differentiation. Thus, two genomic profiles were obtained by ERIC-PCR. The first profile consisted of A. salmonicida subsp. salmonicida strains isolated from the different hosts. The second profile was composed of two A. salmonicida subsp. masoucida and one A. salmonicida subsp. achromogenes. Using REP-PCR, three genotypes were obtained within this pathogen that were related to the diverse subspecies analyzed. In summary, both methodologies are useful for typing distinct strains associated with different host species and therefore are helpful in epidemiological studies of P. anguilliseptica. In contrast, in the case of A. salmonicida, more studies are needed to determine their utility in discriminating the subspecies salmonicida from the other two subspecies.     

Immersion booster vaccination effect on sea bass (Dicentrarchus labrax L.) juveniles

In each challenge 30 sea bass juveniles (mean weight 3.3 +/- 0.2 g SD) were used. During the whole experiment (water T: 18 +/- 1 degrees C) the fish were held in four 50l seawater independent recirculation systems (one fish group per 50l system). The protection to the pathogen Vibrio anguillarum was tested on booster vaccinated sea bass (Dicentrarchus labrax L.) juveniles. The vaccination was performed by immersion for 60 s in a commercial anti-V. anguillarum vaccine suspension. Booster vaccination took place 60 days after the initial immunization. Thirty days after the booster vaccination all the fish received intraperitoneally (IP) 3.0 x 10(6) cfu/fish (colony forming units) virulent V. anguillarum bacteria. The booster vaccination showed a strong protection effect on the challenged sea bass. In the next 20 days after the challenge the mortality was 0% among the booster vaccinated sea bass, 10% among the once vaccinated fish and 50% in the control group (unvaccinated fish). No mortality was observed among the unvaccinated sea bass injected IP with sterile normal saline by the challenge.     

Gill morphology in two Mediterranean Sea fishes of similar feeding preferences: sea bream (Sparus aurata L) and sea bass (Dicentrarchus labrax)

The aim of the present study was to describe the gill morphology of the gilthead sea bream and sea bass fishes that showed the same feeding habits. Our study focused on the gross morphological, scanning and light microscopical features of the gills, where a many differences in the gill arches and gill rakers surface in both species were explained. Our results marked that; there was an interbranchial median elevation appeared in sea bream. The gill arches length and the gaps between decreased medial wards in both species. The gill rakers had similar arrangement and varied number in both species. The first lateral rakers row was the longest one in sea bass. However the gill filaments appeared in double rows. Gill filaments were long at middle and short at extremities of gill arch in both species. Scanning electron microscopy revealed the surfaces of gill arch covered by a mosaic of pavement cells, varied sized pores of chloride cells and many pointed spines on the rakers ventral border in both species. Two types of taste buds marked on the gill arches and rakers in sea bass. Many lamellae appeared on filaments in both species.     

Characterization of the probiotic strain Vagococcus fluvialis in the protection of European sea bass (Dicentrarchus labrax) against vibriosis by Vibrio anguillarum

Aquaculture is one of the main sources of income in many countries worldwide. Intensive farms are often affected by different infectious diseases that can decrease their final production. To control this situation, several antibiotics are frequently used with known environmental consequences. The aim of this study was to analyze different bacterial strains isolated from of gilthead sea bream, sea bass, sole and meagre guts, for use as probiotics in aquaculture. The strains were evaluated in vitro through various mechanisms of selection, such as the production of antagonistic effects against pathogens, production of antibacterial substance, adhesion to the intestinal mucus, competition for nutrients or binding site, and growth in intestinal mucus. A total of 50 bacterial strains were analyzed and only one showed excellent in vitro results for consideration as a candidate to be analyzed in vivo. The strain, identified as Vagococcus fluvialis, showed good protection against Vibrio anguillarum 975-1 in vivo in the experimental challenge, showing a relative percent survival of 42.3% higher than positive control group. Therefore, in conclusion we consider this strain to be a good candidate for use as a future probiotic in aquaculture.     

Unilateral eye abnormalities in reared Mediterranean gilthead sea bream

Eye abnormalities in reared gilthead sea bream (Sparus aurata) were investigated clinically and by histological techniques. A significant number of fish had a cataract in one eye and ocular inflammation including gross exophthalmos. In a small number of fish gas bubbles were observed in the anterior chamber of the eye. Histological examination provided no evidence of an infectious process and a possible behavioural aetiology is discussed.     

Insulin,IGF-I,and muscle MAPK pathway responses after sustained exercise and their contribution to growth and lipid metabolism regulation in gilthead sea bream

Herein, we studied whether sustained exercise positively affects growth of gilthead sea bream by alterations in a) plasma concentrations of insulin and IGF-I, b) signaling pathways in muscle, or c) regulation of lipid metabolism. Specifically, we evaluated the effects of moderated swimming (1.5 body lengths per second; BL/s) on the circulating concentrations of insulin and IGF-I, morphometric parameters, and expression of genes related to lipid metabolism in gilthead sea bream (80–90 g BW). Exercise increased the specific growth rate (P < 0.05) and reduced the hepatosomatic index (P = 0.006). Plasma IGF-I concentrations increased in exercised fish (P = 0.037), suggesting a role for this endocrine factor in the control of muscular growth and metabolic homeostasis during swimming. The observed decrease in plasma insulin concentrations (P = 0.016) could favor the mobilization of tissue reserves in exercised fish. In this sense, the increase in liver fatty acid content (P = 0.041) and the changes in expression of peroxisome proliferator-activated receptors PPARα (P = 0.017) and PPARγ (P = 0.033) indicated a hepatic lipid mobilization. Concentration of glycogen in both white and red muscles was decreased (P = 0.021 and P = 0.017, respectively) in exercised (n = 12) relative to control (n = 12) gilthead sea bream, whereas concentrations of glucose (P = 0.016) and lactate (P = 0.0007) were decreased only in red muscle, indicating the use of these substrates. No changes in the glucose transporter and in lipoprotein lipase mRNA expression were found in any of the tissues studied. Exercised sea bream had decreased content of PPARβ mRNA in white and red muscle relative to control sea bream expression (P = 0.001 and P = 0.049, respectively). Mitogen-activated protein kinase phosphorylation was significantly down-regulated in both white and red muscles of exercised sea bream (P = 0.0374 and P = 0.0371, respectively). Tumor necrosis factor-α expression of white muscle was down-regulated in exercised gilthead sea bream (P = 0.045). Collectively, these results contribute to the knowledge base about hormonal regulation of growth and lipid metabolism in exercised gilthead sea bream.     

The Post-larval Development of Lateral Musculature in Gilthead Sea Bream Sparus aurata (L.) and Sea Bass Dicentrarchus labrax (L.)

Fibre-type differentiation of lateral musculature has been studied in gilthead sea bream Sparus aurata (L.) and sea bass Dicentrarchus labrax (L.) during post-larval development using ultrastructural, histochemical and morphometric techniques. The study showed three muscle layers: red, intermediate (or pink) and white. Initially, most of the red muscle showed low myosin ATPase (m-ATPase) activity fibres, whereas near the transverse septum some small high m-ATPase activity fibres appeared and later acquired a rosette aspect. Afterwards, during adult growth the red muscle showed a histochemical mosaic appearance. The pink muscle in sea bass was observed at the beginning of juvenile development by the oxidative technique (NADH-RT reaction) whereas in gilthead sea bream it was also observed at the end of larval development. The pink layer consists of high m-ATPase activity fibres. However, along the muscle development other low and moderate m-ATPase activity fibres were observed close to the red and white muscles, respectively. The white muscle of juvenile fish showed a histochemical mosaic appearance near the pink muscle. In adult specimens the mosaic white muscle spread out occupying the whole of the myotome. Morphometric analysis shows a significant increase in mean fibre diameter during post-larval development, as shown by the Student's t-test (hypertrophic growth). Skewness and kurtosis values of fibre diameters point to the generation of a new fibres from the myosatellite cells (hyperplastic growth).     

Ozone disinfection of eggs from gilthead seabream Sparus aurata, sea bass Dicentrarchus labrax, red porgy, and common dentex Dentex dentex

The risk of fish pathogen transmission via eggs can be reduced by disinfection in ozonated seawater. The aim of this study was to determine the suitable conditions for ozone disinfection of the eggs of gilthead seabream Sparus aurata, sea bass Dicentrarchus labrax, red porgy Pagrus pagrus, and common dentex Dentex dentex. The eggs were disinfected with a concentration (C) of 0.5 mg of ozone/L of water at four different exposure times (T = 2, 4, 8, and 16 min). The hatching rate was determined in triplicate for each treatment. Bacterial colonies were counted on tryptic soy agar and thiosulfate-citrate-bile salts-sucrose agar. At the end of the experiment, bacterial load and hatching rate were assessed together to determine the optimal ozone treatment values, which were estimated in CT units (i.e., C [= 0.5 mg/L] x T [min]). Optimal values were CT 2-4 (T = 4-8 min; 18 degrees C) for gilthead seabream and red porgy, CT 2 (T = 4 min; 18 degrees C) for common dentex, and CT 4 (T = 8 min; 15 degrees C) for sea bass.     

Viral encephalopathy and retinopathy of Dicentrarchus labrax and Sparus aurata farmed in Tunisia

Viruses belonging to the Nodaviridae family cause disease worldwide among a large number of species of marine fish, and have been described in all continents. In the present study, a total of 69 farmed Tunisian sea bass (Dicentrarchus labrax) and 24 sea bream (Sparus aurata) samples were tested monthly for the detection of betanodavirus. The virus was identified in both species using indirect immunofluorescence assays (IFAT) and RT-PCR. In addition sequence analysis of part of the coat protein gene indicated that both species were infected by highly related, but distinct, strains belonging to the RGNNV genotype. The sequence of the coat protein gene of several strains was identical but up to 9 different sequences were detected in a single farm. In addition, viral sequences obtained from fish that were held at lower temperature (<20°C) were distinct from the rest of the sequences.     

Electron spin resonance (ESR) detection of irradiated fish containing bone (gilthead sea bream,cod, and swordfish)

Food irradiation is a preservation method that has been approved by the European Union for a limited number of foods or food products and within a limited range of doses. European Community legislation states that all foods or food ingredients treated in this manner and authorized for sale in the European Union must be clearly labeled, and that market-level inspections must be carried out to ensure compliance with this regulation. To improve detection of irradiated foods, we evaluated the efficacy of electron spin resonance (ESR) spectroscopy for the identification of seafood that had been subjected to irradiation. Three fish species were tested: gilthead sea bream, cod, and swordfish. For each species, 18 samples of flesh-containing bone were analyzed after either irradiation at 0.5, 1, or 3 kGy or no irradiation (control). Induced radicals in irradiated samples produced distinct ESR signals that allowed differentiation from non-irradiated samples. Within the dose range tested, a linear dose-response relationship was observed (R ² > 0.94). The gilthead sea bream displayed the highest radiosensitivity. The analytical procedure was validated and successfully used for the routine monitoring of locally marketed seafood. The main aims of the study were to develop a reliable tool for the enforcement of the proper labeling of irradiated foods and to obtain preliminary data on compliance with current laws in the Italian market.

     

Characterization and endocrine regulation of proliferation and differentiation of primary cultured preadipocytes from gilthead sea bream (Sparus aurata)

A preadipocyte primary cell culture was established to gain knowledge about adipose tissue development in gilthead sea bream (Sparus aurata), one of the most extensively produced marine aquaculture species in the Mediterranean. The preadipocytes obtained from the stromal-vascular cell fraction of adipose tissue proliferated in culture, reaching confluence around day 8. At that time, the addition of an adipogenic medium promoted differentiation of the cells into mature adipocytes, which showed an enlarged cytoplasm filled with lipid droplets. First, cell proliferation and differentiation were analyzed under control and adipogenic conditions during culture development. Next, the effects of insulin, GH, and IGF-I on cell proliferation were evaluated at day 8. All peptides significantly stimulated proliferation of the cells after 48 h of incubation (P < 0.002 for GH and IGF-I and P < 0.05 for insulin), despite no differences were observed between the different doses tested. Subsequently, the effects of insulin and IGF-I maintaining differentiation when added to growth medium were studied at day 11, after 3 d of induction with adipogenic medium. The results showed that IGF-I is more potent than insulin enhancing differentiation (P < 0.01 for IGF-I compared with the control). In summary, a primary culture of gilthead sea bream preadipocytes has been characterized and the effects of several regulators of growth and development have been evaluated. This cellular system can be a good model to study the process of adipogenesis in fish, which may help improve the quality of the product in aquaculture.     

水貂沙门氏菌的分离鉴定及药物敏感性分析

为指导临床合理用药并为治疗患病水貂提供依据,本试验对山东省某水貂养殖场送检的7只疑似患有沙门氏菌病的病死水貂的肝脏和脾脏样品进行细菌分离鉴定及药物敏感性分析,通过分离纯化方法从样品中分离菌株,并采用革兰氏染色、生化鉴定和PCR方法对分离菌株进行鉴定。运用K-B药敏法检测菌株对临床常用药物的敏感性,并通过PCR方法检测菌株耐药基因及Ⅰ类整合子的携带情况。结果显示,本试验分离得到的7株菌均为革兰氏阴性、短小的杆菌;生化反应检测显示,分离菌株葡萄糖、麦芽糖、甘露醇、MR试验、枸橼酸盐、硫化氢试验均表现为阳性,初步鉴定分离菌株为沙门氏菌;PCR产物测序结果进一步表明7株分离菌均为沙门氏菌;药敏试验结果显示,7株沙门氏菌对头孢曲松、左氧氟沙星、氟苯尼考和多黏菌素较敏感,对氨基糖苷类药物、四环素和氨苄西林表现为耐药;耐药基因检测结果显示,7株沙门氏菌共检测出8种耐药基因blaTEM-1、blaCTX-M-1G、aadA1、aac(3′)-Ⅳ、aac(3′)-Ⅱc、aph(4′)-Ⅰa、aph(3′)-Ⅶ和oqxAB,以及基因盒为aadA1、arr-3-aacA4和blaPSE-1的Ⅰ类整合子。综上可知,本试验在送检的7只患病水貂的肝脏和脾脏样品中分离到7株沙门氏菌,分离菌株均为耐药菌株,且主要表现为多重耐药现象;耐药基因呈多样性,且存在位于质粒上的耐药基因扩大了耐药基因的传播范围,增加了细菌的耐药性,为临床用药治疗带来困难。     

Drug resistance in strains of Escherichia coli isolated from the intestinal tract of pigs in Norway.

Faecal samples from 95 healthy pigs and samples of jejunal content from 85 piglets suffering from colienterotoxaemia were tested for the presence of drug resistant E. coli strains. Practically all pigs in both groups harboured E. coli strains resistant to one or more of the 6 antibiotics/chemotherapeutic agents tested (Oxytetracycline, streptomycin, sulphaisodimidin, neomycin, ampicillin, chloramphenicol). Almost 100% of healthy and approx. 90% of diseased pigs harboured strains resistant to Oxytetracycline, streptomycin and sulphaisodimidin. Pigs with strains resistant to neomycin, ampicillin and chloramphenicol were less frequently found. The predominant coliform flora consisted of E. coli strains” resistant to Oxytetracycline, streptomycin and sulphaisodimidin in 71% to 81% of diseased pigs and in 47% to 69% of the healthy pigs. In diseased pigs ¾ of the animals had a coliform flora dominated by neomycinresistant E. coli strains.Of the 721 resistant E. coli strains isolated from healthy pigs, 11% were single resistant while the corresponding figure for the 518 resistant strains isolated from diseased pigs was 6%. Thus 89% and 94% of strains showed simultaneous resistance to 2 or more antibiotics. E. coli strains resistant to 3 or more drugs were found in approx. 60% and 70% of the isolates from healthy and diseased animals, respectively. Oxytetracycline/streptomycin/sulphaisodimidin resistance was most commonly found, approx. 22% and 38% of the strains from healthy and diseased pigs, respectively, showing this resistance pattern.Transmission of drug resistance which was examined in E. coli strains originating from the diseased pigs was demonstrated in approx. 76% of the isolates. The incidence of drug resistance transfer in single, double, triple and quadruple resistant strains was 11%, 68%, 97% and 98%, respectively.     

Interactions between bacteria and Cryptosporidium molnari in gilthead sea bream (Sparus aurata) under farm and laboratory conditions

The possible interaction of Cryptosporidium molnari and bacteria in gilthead sea bream (Sparus aurata) was studied. Epidemiological data from a pathological survey under farm conditions were analyzed. In addition, parasite and bacteria burdens were studied in experimental models in which naturally and experimentally parasitized fish were challenged with a particular strain of Vibrio harveyi (H57). All the bacteria species present were studied. Under farm conditions, the parasite was more prevalent when mortality or morbidity cases (study C) occurred than in randomly sampled fish (study B). In study C, parasite abundance was significantly higher in bacteria-negative fish, and total bacteria abundance was significantly higher within non-parasitized fish. V. harveyi and V. splendidus were the most prevalent among bacteria carriers in studies B and C, respectively. In study C, among bacteria carriers, most isolates were slightly more prevalent in parasitized than in non-parasitized fish. Two groups (G1, G2) of naturally parasitized fish were inoculated with H57 by intracoelomic injection (ICI) and by oral intubation (OI). H57 was recovered only in G1 inoculated fish, which had a significantly higher basal abundance of total bacteria, and where the only ones with mortalities. In G1, the mortality rate and the prevalence of other V. harveyi strains different from the H57 molecular type were higher in ICI than in OI fish, and the total bacteria abundance was also significantly higher in ICI fish. C. molnari abundance was significantly higher in G1 than in G2, and also in OI than in ICI fish within G1. When H57 was IC inoculated to fish (G3, from the same farm as G2) experimentally infected with C. molnari, H57 was not recovered from any fish. A low mortality was recorded, and only in those fish inoculated with both pathogens. Also in these fish, the prevalence of infection of C. molnari was higher and histopathological damage to the stomach was greater than in fish inoculated only with the parasite. Therefore, the impact of the parasite would be reduced notably when the bacterial burden or the intensity of parasite infection are low (G2, G3).     

Review on the immunology of European sea bass Dicentrarchus labrax

European sea bass (Dicentrarchus labrax L.) is a marine species of great economic importance, particularly in Mediterranean aquaculture. However, numerous pathogenic viruses, bacteria, fungi and parasites affect the species, causing various infectious diseases and thereby leading to the most heavy losses in aquaculture production of sea bass. In this respect, knowledge on molecular and genetic mechanisms of resistance to pathogens and specific features of immune response against various infectious agents should greatly benefit the development of effective vaccines and proper vaccination strategies in marker-assisted selection of fish resistant to a range of infections. To date, genetic knowledge on sea bass immune regulatory genes responsible for resistance to pathogens is relatively poor but tends to accumulate rapidly. In this review, we summarize and update current knowledge on the immune system and immune regulatory genes of the sea bass.     

Preliminary assessment of dietary mannanoligosaccharides on growth performance and health status of gilthead seabream Sparus auratus

A feeding trial was performed to assess the potential beneficial effect of two levels of mannanoligosaccarides (MOS) on the growth performance, feed utilization, hematological parameters, and liver histopathology of gilthead seabream Sparus auratus (also known as gilthead bream). Mannanoligosaccarides were added at the rates of 2 and 4 g/kg to a fish-meal-based control diet, and each diet was given (twice daily [midmorning and midafternoon] to apparent satiation) to triplicate groups of gilthead seabream growers (mean weight = approximately 170 g) in sea cages. The trial lasted 12 weeks, and the average ambient water temperature ranged from 19.6 degrees C to 24.7 degrees C during the experimental period. At the end of the experiment, fish attained market size (350-450 g) and their health status was evaluated by blood analysis and liver histology. There were no differences in survival rates among fish fed experimental diets. However, there were significant improvements in both growth and feed utilization among fish fed diets supplemented with MOS. Hemoglobin (Hb) and hematocrit (Ht) levels and erythrocyte, leukocyte, and thrombocyte (Thr) counts were unaffected by any dietary MOS. The levels of Hb (g/dL; mean +/- SD) and Ht (%; mean +/- SD) were 11.0 +/- 2.5 and 45.6 +/- 6.7 for the control group, 11.1 +/- 1.7 and 39.3 +/- 8.0 for the 2-g/kg group, and 11.2 +/- 1.9 and 40.2 +/- 8.4 for the 4-g/kg group. The mean Thr count ranged from 47.6 to 53.8 x 10(3)/mm3. Despite the apparently higher Thr counts for fish fed diets supplemented with MOS, these differences were not significant. Moreover, no histopathological differences were observed in liver tissue cross sections between control and treatment groups. These results suggest that supplementation of diets with MOS had no significant effects on general fish health.     

重庆部分羊场沙门氏菌的分离鉴定、耐药表型分析及耐药基因检测

本研究旨在初步了解重庆市羊源沙门氏菌的耐药性及耐药基因流行情况。在5个山羊养殖场采集185份山羊粪便样品，经选择性增菌和PCR鉴定分离沙门氏菌，确定了其血清型，测定了分离菌对28种抗菌药物的敏感性，并检测了喹诺酮耐药决定区（QRDR）的耐药突变位点和质粒介导的喹诺酮耐药（PMQR）、超广谱β-内酰胺酶（ESBL）基因。共分离到羊源沙门氏菌11株，其中10株为德尔卑沙门氏菌。分离的菌株对氨苄西林、头孢唑啉、头孢氨苄、头孢噻肟、四环素和培氟沙星耐药严重；9株菌表现为多重耐药，对3~7类药物耐药。所有菌株均存在QRDR耐药突变且携带bla_TEM基因；7株菌携带1~5种PMQR基因。本研究分离的羊源沙门氏菌对常用抗菌药物整体耐药较为严重，且广泛存在耐药突变和携带耐药基因。     

一株产志贺毒素Ⅱ且山梨醇阳性的大肠杆菌O157：H7的分离与鉴定

在上海某猪场采集6份腹泻猪粪便样品和2份病死猪小肠内容物样品,增菌后经麦康凯培养基和伊红美兰培养基分离得到32株疑似大肠杆菌,通过PCR方法分析O157特异基因rfbE和血清学鉴定,分离获得一株肠出血性大肠杆菌O157：H7。PCR检测该菌株的毒力因子携带情况,结果表明其只携带志贺毒素Ⅱ,而不携带志贺毒素I、紧密素和溶血素等毒力因子;生化试验发现该菌株与大多数O157：H7特性不同,可分解山梨醇;毒力试验和耐药性试验表明该菌株毒性较强,且耐药性很强,对大多数抗生素不敏感。     

Genomic relatedness among Actinobacillus pleuropneumoniae field strains of sterotypes 1 and 5 isolated from healthy and diseased pigs.

Forty-four Actinobacillus pleuropneumoniae isolates recovered from both healthy and diseased pigs were characterized by random amplified polymorphic DNA analysis (RAPD), pulsed field gel electrophoresis (PFGE) and apx toxin gene typing. Nine RAPD types and 14 PFGE patterns were identified. No common RAPD or PFGE patterns were found between strains of serotype 1 and those of serotype 5. The RAPD analysis indicated that the 15 serotype 1 strains isolated from diseased pigs were assigned to 4 RAPD types, with 66% of strains characterized by the same RAPD type. By contrast, the 5 strains of serotype 1 isolated from healthy carriers were dispersed in 4 RAPD types. These data suggest that the diversity of strains isolated from healthy pigs could be higher than that of strains recovered from diseased pigs. In addition, all serotype 5 strains exhibited a unique RAPD type. Unlike RAPD, PFGE analysis allowed discrimination among isolates of serotype 1 and among those of serotype 5. All but 3 isolates showed the same apx genotype as their respective serotype reference strain. These data indicate that RAPD analysis is a valuable rapid tool for routine subtyping of strains of serotype 1. For strains of serotype 5, a combination of several typing methods, such as PFGE and apx gene typing, is needed to provide useful information on the molecular epidemiology of swine pleuropneumonia.