The efficacy of a Leptospira interrogans serovars pomona and copenhageni and L. borgpetersenii serovar hardjo vaccine in cattle

An experimental, trivalent, bovine, leptospiral vaccine, containing inactivated Leptospira interrogans serovars pomona and copenhageni and L. borgpetersenii serovar hardjo Hardjobovis, was developed. The experimental vaccine was shown to protect hamsters against virulent challenge with each of the component serovars. In a serological efficacy test in cattle, the experimental vaccine was compared for bioequivalence with a similar product, registered in New Zealand for veterinary use. The experimental vaccine induced higher titres in cattle than the latter mentioned product.     

Dual infections of red deer (Cervus elaphus) by Leptospira interrogans serovars copenhageni and hardjo

On a property in the Nelson District, blood and urine samples were taken from red deer (Cervus elaphus) from which low (<1:100) antibody titres to serovar copenhageni and suspected leptospiral abortions had previously been reported. A total of 27 hinds were sampled. Microscopic agglutination test (MAT) titres in sera ranging from 1:32 to 1:128 were found in six animals. Thirteen leptospiral isolations were made from nine of the 27 urine samples. Four of these were typed as copenhageni and nine as hardjo. Two cultures were prepared from each urine sample and hardjo and copenhageni were both isolated from single urine samples from two animals. None of the 27 deer had serum MAT titres at 1:32 or above to copenhageni.     

Antibodies in dogs against Leptospira interrogans serovars copenhageni, ballum and canicola

In a nationwide survey carried out during 1990-91 of more than 5800 dogs to detect antibodies against Leptospira interrogans serovars copenhageni, ballum and canicola, only one weak reactor against serovar canicola was found. Reactors of varying titre were found against serovar ballum in 0.7% of dogs tested, indicating sporadic infection with this serovar. Reactors (0.9%) to serovar copenhageni came mainly from the Waikato, Northland and the Auckland region. This was in agreement with the reported occurrence of the clinical syndrome and with the results of a smaller survey in urban Auckland, in which more than 5% of dogs tested were seropositive to serovar copenhageni.     

A serosurvey for antibodies to Leptospira in dogs in the lower North Island of New Zealand

AIMS: To investigate the prevalence of antibodies to endemic and exotic Leptospira serovars in samples from a serum bank, collected from dogs in the lower North Island of New Zealand. METHODS: Sera (n=466), which had been collected from apparently healthy dogs, were screened using the microscopic agglutination test (MAT) for antibodies to serovars L. borgpeterseni serovar hardjo, L. interrogans serovars pomona, copenhageni and canicola, and L. kirschneri serovar grippotyphosa. RESULTS: Antibody to Leptospiral antigen was found in 14.2% of dogs tested. The highest level of reactivity was with serovar copenhageni, to which 9.5% (41/433) of sera were positive. Antibodies to serovars grippotyphosa and canicola were not detected in this population of dogs. CONCLUSIONS: Leptospira infection is relatively common in dogs in the lower North Island . CLINICAL RELEVANCE: Vaccination of dogs against leptospirosis should be considered using vaccine containing antigen to serovars hardjo, pomona and copenhageni. The presence of moderate levels of copenhageni antibody in dogs in the lower North Island raises the possibility that this serovar has become established in rodent populations in this region.     

Reorganisation of the caecal extracellular matrix upon Salmonella infection--relation between bacterial invasiveness and expression of virulence genes

Interactions of Salmonella (S.) outer membrane structures with extracellular matrix (ECM) of host tissues seem to be crucial for bacterial adhesion and invasion. To evaluate the relationship between the ECM and bacterial invasiveness, the reorganisation of fibronectin, tenascin-C and laminin after Salmonella exposure in vivo, the Salmonella adhesiveness to ECM proteins in vitro and the virulence gene expression upon co-cultivation of salmonellae and ECM proteins were elucidated for two Salmonella strains with different capabilities to enter the intestinal mucosa. Immunohistochemistry and confocal microscopy showed that the infection of day-old chicks using either the highly invasive S. Enteritidis (SE) or the nearly non-invasive S. Infantis (SINF) strain was associated with an invasion-dependent reorganisation of fibronectin and tenascin-C in the caecal wall. Compared to SINF, clustered formations of SE were localised within and attached to the fibronectin and tenascin-C scaffold in the lamina propria indicating a relevance of ECM for bacterial dissemination in lower regions of the mucosa. In adhesion assays, SE was, indeed, significantly more adhesive to the matrix proteins than SINF. The attachment was accompanied by an increased fliC mRNA expression in SE demonstrated by microarray analysis as well as quantitative real-time RT-PCR. The data suggest a relationship between the capability of Salmonella serovars to interact with matrix proteins and to disseminate in gut mucosa perhaps in consequence of a matrix-mediated upregulation of the Salmonella motility gene fliC.     

Potency of leptospiral vaccines and protection against chronic infection in golden hamsters

Seven vaccines prepared from pathogenic strains of different origin of Leptospira interrogans [serovars icterohaemorrhagiae (one strain) and copenhageni (6 strains)] were examined in protection tests on golden hamsters. Two of the copenhageni strains were used for challenge. The organs (kidneys, spleen, liver) in the vaccinated animals surviving challenge were protected to a varying degree. Low rates of survival were associated with a high incidence of Leptospira-positive findings, partly connected with focal lesions of the kidneys. On the other hand, in the groups in which all the animals survived, it was not possible to culture leptospires from their organs or to detect leptospiral antigen in these organs by immunohistochemical investigation. A protection of the organs that prevents vaccinated animals from shedding leptospires after infection clearly depends on the vaccine dose administered and the efficacy of the vaccine which can be measured in potency tests based on the survival rate as the relevant parameter.     

Factors associated with virulence of Mycoplasma synoviae.

Virulence mechanisms of six isolates of Mycoplasma synoviae (MS), previously classified as pathogenic (K1968), moderately pathogenic (WVU 1853, K1858, 92D8034, and F10-2AS), and mildly pathogenic (FMT) in chickens, were examined. The most virulent isolate, K1968, had been found to invade systematically and produce lesions following eye-drop inoculation. In the present study, all isolates were evaluated for presence of a possible cytadhesin and for functional attachment to host cells as indicated by hemagglutination and hemadsorption. Three representative isolates, K1968, 92D8034, and FMT, were evaluated for attachment and colonization in cultured chick tracheal rings and tendon cell monolayers by direct transmission electron microscopic examination and by quantitative polymerase chain reaction assay. Ciliostasis was compared in tracheal organ culture. Previously found differences in pathogenicity of these isolates for chickens could not be explained as differences in attachment and were only partially explained by differences in colonization. Pathogenicity of the most virulent isolate of MS was suspected to be multifactorial, involving attachment and colonization of the upper respiratory tract plus additional unidentified factors associated with systemic invasion and lesion production.     

Detection of virulent strains of Streptococcus suis type 2 and highly virulent strains of Streptococcus suis type 1 in tonsillar specimens of pigs by PCR.

We developed a PCR assay for the rapid and sensitive detection of virulent Streptococcus suis type 2 and highly virulent S. suis type 1 in tonsillar specimens from pigs. The PCR primers were based on the sequence of the gene encoding the EF-protein of virulent S. suis type 2 strains (MRP+EF+) and highly virulent S. suis type 1 strains (MRP(s)EF+) and of the EF protein of weakly virulent S. suis type 2 strains (MRP+EF). The latter strains give rise to larger PCR products than the virulent strains of S. suis type 1 and 2. A positive control template was included in the assay to identify false negative results. The PCR was evaluated using tonsillar specimens from herds known (or suspected) to be infected and herds without an S. suis history. The results obtained with the PCR assay were compared with the results obtained with a newly developed bacteriological examination. In this bacteriological examination we were able to identify the EF-positive strains directly in the tonsillar specimens. From the 99 tonsils examined, 48 were positive in the PCR and 51 negative. All specimens from which EF-positive S. suis strains were isolated were also positive in the PCR assay. Three samples were positive in the PCR, but negative by bacteriological examination. The results demonstrated that the PCR is a highly specific and sensitive diagnostic tool for the detection of pigs carrying virulent strains of S. suis type 2 and highly virulent strains of type 1. Application of the assay may contribute to the control of S. suis infections.     

Interactions of highly and low virulent Mycoplasma hyopneumoniae isolates with the respiratory tract of pigs

Mycoplasma hyopneumoniae is the etiological agent of swine enzootic pneumonia, a chronic nonfatal disease affecting pigs of all ages. To obtain better insight in the mechanisms responsible for differences in virulence between highly and low virulent M. hyopneumoniae isolates, 23 caesarean-derived, colostrum-deprived piglets were randomly assigned to three groups. Groups 1 and 2 consisted of nine animals each, which were intratracheally inoculated at 1 week of age with a highly or a low virulent isolate of M. hyopneumoniae, respectively. The remaining five animals were inoculated with sterile culture medium. Animals were euthanized at 5, 10, 15 and 28 days post-inoculation (DPI). Animals inoculated with the highly virulent isolate had more neutrophils in BAL fluid at 10, 15 and 28DPI compared to the other groups. At 10 and 15DPI, animals in the highly virulent group had significantly higher concentrations of TNF-alpha in BAL fluid. IL-1beta concentration in this group was higher at 5 and 28DPI compared to the other groups. From 10DPI onwards, significantly higher titres of M. hyopneumoniae were detected in the BAL fluid of animals inoculated with the highly virulent isolate compared to animals inoculated with the low virulent isolate. Additionally, the in vitro generation time of the highly virulent M. hyopneumoniae isolate was significantly shorter than that of the low virulent isolate. The present study indicates that the difference in pathogenicity between the highly and low virulent isolates is associated with a faster in vitro growth, a higher capacity to multiply in the lungs and the induction of a more severe inflammation process by the highly virulent isolate.     

Biological properties of allogenic articular chondrocytes on the surface of bovine cartilage explants in vitro

Bovine cartilage explants were co-cultured with or without allogenic chondrocytes for 4 weeks. The attachment of the applied chondrocytes to cartilage after labelling with fluorescence was assessed using a confocal laser microscope. Morphological changes and the production of extracellular matrix (ECM) of co-cultured chondrocytes on intact and damaged surfaces of cartilage were evaluated by histological and immunohistochemical methods. Co-cultured chondrocytes attached to and proliferated on the intact and damaged areas of cartilage, and a new layer was created there. The defects were also filled with ECM produced by the co-cultured chondrocytes. Glycosaminoglycans and collagen type II were detected in the newly formed ECM, and large numbers of rounded chondrocytes were observed at primitive lacunae in this matrix at 4 weeks of culture. The results suggest that chondrocytes have the ability to attach to, to proliferate on and to establish a new matrix on the intact and damaged surfaces of cartilage explants.     

Virulence of avian capsular serogroup B Pasteurella multocida for turkey poults

Two strains of capsular serogroup B Pasteurella multocida isolated from avian hosts (swan and turkey) were evaluated for virulence based on lethality for turkey poults. Groups of poults were exposed intramuscularly to various concentrations of organisms of each strain. Both strains were virulent. The strain isolated from a turkey was highly virulent: all exposed poults died in less than 24 hours, including those exposed to only 79 organisms. This highly virulent strain was neither highly invasive nor highly infective: intrapharyngeal exposure with 7.9 x 10(6) organisms resulted in death of only one of five poults, and attempts to isolate the organism from pharyngeal mucosae and livers of surviving poults were unsuccessful. The high degree of virulence of a B capsular group strain isolated from a turkey indicates a disease-producing potential for members of this uncommon serogroup of P. multocida.     

Serological survey of leptospirosis in sows with premature birth and stillbirth in Chiba and Gunma prefectures of Japan

In 1996 and 1997, the seroprevalence against Leptospira in parturient sows with premature birth or stillbirth from two herds was investigated. In three out of four sow serum samples obtained in Gunma Prefecture, the antibody titers to Leptospira interrogans serovar copenhageni (M20) were higher than 10,000 (the reciprocal of the serum dilution). Furthermore, the antibody titers to L. interrogans serovar canicola (Hond Utrecht IV) were significantly high in the three sows and the titers ranged from 1,000 to 3,000. In sows obtained in Chiba Prefecture, significantly high antibody titers to serovar copenhageni (M20) were confirmed in eight out of 40 sows, and antibody titers greater than 10,000 in six of them. Significantly high antibody titers to L. interrogans serovar icterohaemorrhagiae (RGA) and L. canicola (Hond Utrecht IV) were confirmed in four and 18 out of the 40 sows, respectively, compared with the titers to the other serovars. These findings may indicate the prevalence of leptospirosis in pig herds in both Gunma and Chiba Prefectures.     

Identification of unique DNA sequences present in highly virulent 2009 Alabama isolates of Aeromonas hydrophila

In 2009, a disease outbreak caused by Aeromonas hydrophila occurred in 48 catfish farms in West Alabama, causing an estimated loss of more than 3 million pounds of food size channel catfish. Virulence studies have revealed that the 2009 isolates of A. hydrophila are at least 200-fold more virulent than a 1998 Alabama isolate AL98-C1B. However, up to now, no molecular markers have been identified to differentiate the highly virulent 2009 isolates from other isolates of A. hydrophila. To understand the genetic differences between the highly virulent 2009 isolates and the less virulent AL98-C1B at molecular level, PCR-select bacterial genome subtractive hybridization was used in this study. A total of 96 clones were selected from the subtractive genomic DNA library. Sequencing results revealed that the 96 clones represented 64 unique A. hydrophila sequences. Of the 64 sequences, three (hypothetical protein XAUC_13870, structural toxin protein RtxA, and putative methyltransferase) were confirmed to be present in the three virulent 2009 Alabama isolates but absent in the less virulent AL98-C1B. Using genomic DNAs from nine field isolates of A. hydrophila with different virulence as templates, two sequences (hypothetical protein XAUC_13870 and putative methyltransferase) were found to be only present in highly virulent A. hydrophila isolates, but absent in avirulent isolates.     

Experimentally induced leptospirosis in coyotes (Canis latrans)

Infection of coyotes (Canis latrans) with Leptospira interrogans serovars pomona, canicola, and copenhageni was accomplished by percutaneous inoculation. Bacteriologic, serologic, histopathologic, and fluorescent antibody techniques were used to investigate the infections. Leptospiremia was established with pomona. Leptospiruria was demonstrated with the three serovars. Serovar canicola was recovered from one coyote 134 days after it was inoculated.     

Meat Science and Muscle Biology Symposium: the influence of extracellular matrix on intramuscular and extramuscular adipogenesis

The extracellular matrix (ECM) and specific ECM components can have a major influence on cell growth, development, and phenotype. The influence of the ECM and ECM components on adipogenesis in vivo and in vitro will be reviewed in this paper. Engelbreth-Holm-Swarm substratum and laminin per se markedly increased attachment, spreading, and hypertrophy of preadipocytes in serum-free primary cultures of porcine adipose tissue stromal-vascular cells. Furthermore, primary cultures of stromal-vascular cells showed that preadipocytes express ECM components after preadipocyte recruitment. Staining for plant lectins, type IV collagen, and laminin in fetal pig adipose tissue demonstrates that adipocyte reactivity for laminin was strong throughout fetal development and was similar for developing adipocytes and vasculature. However, lectin binding and type IV collagen reactivity of blood vessels preceded that for adipocytes. Therefore, these studies indicated that the ECM and in particular laminin may play a critical role in morphological aspects of preadipocyte development. Specific inhibitors and modulators of collagen synthesis have been used to evaluate the role of collagens in the differentiation of bovine intramuscular preadipocytes (BIP) and other preadipocyte cell lines. Triglyceride accretion of BIP cells was inhibited by a general inhibitor of collagen biosynthesis, whereas specific inhibitors or modulators of type IV collagen inhibited 3T3-L1 cell differentiation. Further study revealed that compared with collagens types I to IV, type V and VI collagens have an important and active role in BIP adipogenesis. The growth of intramuscular bovine adipose tissue may be dependent on collagen newly synthesized and organized by the adipocytes per se. The role of extracellular or ECM proteolysis in regulating adipogenesis also will be reviewed in this paper. Many members of the matrix metalloproteinase (MMP) family are expressed by adipocytes, and specific inhibition of MMP-9 greatly reduces adipogenesis in vitro. Possibly, MMP and other proteases regulate turnover of key adipocyte ECM proteins that are involved in the regulation of preadipocyte proliferation and differentiation.     

Presence of the Streptococcus suis suilysin gene and expression of MRP and EF correlates with high virulence in Streptococcus suis type 2 isolates

Nineteen Streptococcus suis type 2 isolates that had been analyzed previously for hemolysin production, ribotype, and virulence in pigs were examined for presence of the gene coding for suilysin by PCR amplification, and southern blot and hybridization techniques. Based on southern blot and hybridization analysis, all isolates tested contained at least a portion of the suilysin gene. PCR amplification of the entire gene resulted in gene fragments from five of the seven highly virulent isolates and none of the moderately virulent or avirulent isolates. Additional PCR analysis showed that mutation or deletions at the 5′ end of the suilysin gene in the less virulent isolates prevented amplification of the sly gene fragment from those isolates. The MRP+ (muramidase-released protein) EF+ (extracellular protein) phenotype was also expressed by the same five highly virulent/sly+ isolates.     

Association of infectivity, parasitaemia and virulence in a serodeme of Trypanosoma congolense

Quantitative methods were adopted to study the course of Trypanosoma congolense infection in mice and goats. The ease of initiating infection with a single organism (clone) was found to show a smooth correlation with the virulence of 24 isolates. Virulence of T. congolense was found to be directly related to the degree of viability of the parasite but inversely proportional to the capacity of the host to limit parasitaemia. Isolates obtained from the goat in the early stage of the infection were found to be highly infective but moderately virulent; subsequent isolates were of low infectivity and low virulence. Organisms obtained at the terminal stage of the infection were highly virulent. Generally, the highly virulent clones produced rapidly high parasitaemia and mortality in the host. The low virulence clones were characterised by low parasitaemia but very pronounced remission of trypanosomaemia.     

Susceptibility of Mongolian gerbils (Meriones unguiculatus) to leptospires and the protective effect of vaccination

Mongolian gerbils, Meriones unguiculatus, were shown to be highly susceptible to Leptospira interrogans serovars such as icterohaemorrhagiae, copenhageni, canicola, autumnalis, javanica, pyrogenes and hebdomadis as compared to guinea pigs and hamsters. Mortality with signs of haemorrhage and jaundice was recorded in all experimental rodents after intraperitoneal inoculation with all strains of the serovars indicated. However, Mongolian gerbils were comparatively susceptible to strains which were of low virulence to guinea pigs and hamsters. Use of leptospiral vaccination proved effective in protecting the animals against inoculum challenges. The Mongolian gerbil is a species that may be selectively and preferentially useful for assays on the protective effects of leptospiral vaccination.     

Genomic fingerprinting of pigeon Streptococcus gallolyticus strains of different virulence by randomly amplified polymorphic DNA (RAPD) analysis

Randomly amplified polymorphic DNA (RAPD) analysis was performed on 95 pigeon S. gallolyticus strains of different virulence and belonging to different biotypes and different culture supernatant phenotypes as determined by SDS-PAGE. Four distinct RAPD patterns, designated A, B, C and D, were distinguished using primer OPM6 (5'CTGGGCAACT). All 76 strains generating RAPD pattern A or B were designated highly virulent on the basis of their SDS-PAGE pattern. Five of seven strains generating RAPD pattern C and 11 of 12 strains generating RAPD pattern D belonged to the moderately virulent and low virulent culture supernatant phenotype groups, respectively. Only one RAPD group C strain belonged to a highly virulent culture supernatant phenotype group. There was a correlation between biotype and RAPD patterns. These findings indicate that there is a high correlation between RAPD pattern and virulence for pigeons. Therefore, RAPD typing seems a rapid, reliable method to distinguish pigeon S. gallolyticus strains of high, moderate and low virulence.