Chao-Fu Chang

Ciprofloxacin-resistant Salmonella enterica Typhimurium and Choleraesuis from Pigs to Humans, Taiwan

We evaluated the disk susceptibility data of 671 nontyphoid Salmonella isolates collected from different parts of Taiwan from March 2001 to August 2001 and 1,261 nontyphoid Salmonella isolates from the National Taiwan University Hospital from 1996 to 2001. Overall, ciprofloxacn resistance was found in 2.7% (18/671) of all nontyphoid Salmonella isolates, in 1.4% (5/347) of Salmonella enterica serotype Typhimurium and in 7.5% (8/107) in S. enterica serotype Choleraesuis nationwide. MICs of six newer fluoroquinolones were determined for the following isolates: 37 isolates of ciprofloxacin-resistant (human) S. enterica Typhimurium (N = 26) and Choleraesuis (N = 11), 10 isolates of ciprofloxacin-susceptible (MIC <1 μg/mL) (human) isolates of these two serotypes, and 15 swine isolates from S. enterica Choleraesuis (N = 13) and Typhmurium (N = 2) with reduced susceptibility to ciprofloxacin (MIC >0.12 μg/mL). Sequence analysis of the gryA, gyrB, parC, parE, and acrR genes, ciprofloxacin accumulation; and genotypes generated by pulsed-field gel electrophoresis with three restriction enzymes (SpeI, XbaI, and BlnI) were performed. All 26 S. enterica Typhimurium isolates from humans and pigs belonged to genotype I. For S. enterica Choleraesuis isolates, 91% (10/11) of human isolates and 54% (7/13) of swine isolates belonged to genotype B. These two genotypes isolates from humans all exhibited a high-level of resistance to ciprofloxacin (MIC 16–64 μg/mL). They had two-base substitutions in the gyrA gene at codons 83 (Ser83Phe) and 87 (Asp87Gly or Asp87Asn) and in the parC gene at codon 80 (Ser80Arg, Ser80Ile, or Ser84Lys). Our investigation documented that not only did these two S. enterica isolates have a high prevalence of ciprofloxacin resistance nationwide but also that some closely related ciprofloxacin-resistant strains are disseminated from pigs to humans.

Immunogenicity and protective efficacy of recombinant Leptospira immunoglobulin-like protein B (rLigB) in a hamster challenge model

Leptospiral immunoglobulin-like protein (LigB) was truncated into conserved (LigBcon) and variable (varB1, varB2) fragments and expressed as GST/His-tag fusion proteins. Four-week-old hamsters were immunized with equal amounts of each fragment individually or combined in alum adjuvant at days 0 and 21 and subsequently challenged three weeks after the booster with 2.5 LD(50) live virulent Leptospira interrogans serovar Pomona. Our results demonstrate that immunization with LigB produced strong humoral immune responses as revealed by high titers against each fragment and significant enhancement in Th2 cytokines (IL-4, IL-10). A significant activation of CMI is revealed by enhanced proliferation of lymphocytes and up regulation of Th1 cytokines (IL-12p40, IFN-gamma) was also noted. Of the peptides studied, rLigBcon was able to impart maximum protection (71%), followed by rVarB1 (54%), whereas rVarB2 was not able to impart a significant level of protection (33%) against lethal infection as revealed by enhanced survival and reduced severity of histopathological lesions in vital organs (viz. kidney, liver, spleen) of the immunized animals. Moreover, concurrent administration of all three fragments significantly enhanced the protective efficacy of the vaccine (83%). Overall, our results clearly demonstrate that LigB has emerged as novel protective antigen that can be used in future subunit vaccines against leptospirosis.

EXPERIMENTAL INFECTION OF THE HUMAN GRANULOCYTIC EHRLICHIOSIS AGENT IN HORSES

Human blood collected from two patients from Westchester County, New York with human granulocytic ehrlichia (HGE) infection was inoculated into two ponies. Inoculated ponies developed clinical signs similar to a previous report (Madigan et al., 1995). Histopathological changes involved follicular hyperplasia of lymphoid tissues. HGE DNA was detected by PCR in muscle, fascia, peritoneum, and adrenal gland after the ponies produced a high level of antibodies to HGE. We suggest that HGE may reside in poorly vascularized connective tissues, where the antibodies may have some difficulties to penetrate, resulting in persistent infection. Since HGE and E. equi cause very similar diseases in both humans and horses, they may be the same organism with minor genetic differences.

Evaluation of immune responses and protective efficacy in a goat model following immunization with a coctail of recombinant antigens and a polyprotein of Mycobacterium avium subsp. paratuberculosis

The protective efficacy of four recombinant antigens (85A, 85B, superoxide dismutase [SOD], and a fusion polypeptide [Map74F]) of Mycobacterium avium subsp. paratuberculosis (MAP) along with the adjuvant dimethydioctadecyl ammonium bromide (DDA) was assessed in a goat challenge model. Animals were immunized with the four antigens with adjuvant DDA (Group I, eight goat kids) or without the adjuvant (Group II, eight goat kids) or adjuvant only (Group III, nine goat kids). Animals were boostered 3 weeks after the primary vaccination and challenged 3 weeks after the booster. Significant antigen-specific lymphoproliferation was observed in the immunized animals 3 weeks after the booster immunization. This response increased further at 4 weeks after the booster. Similarly, antigen-specific IFN-gamma responses increased in the immunized animals 3 weeks after the booster. The response was significantly higher for 85A and Map74F at 10 weeks after primary vaccination (APV) in Group I animals compared to the other two groups. CD4+ T-cell populations were higher in the vaccinated animals from 6 to 10 weeks APV than those of the control animals. A significant increase in recombinant antigen-specific IFN-gamma gene expression was detected in the vaccinated animals. At necropsy (38 weeks APV), our multicomponent subunit vaccine imparted a significant protection in terms of reduction of MAP burden in target organs as compared to sham-immunized goats. This study indicates that our multicomponent subunit vaccine induced a good Th1 response and conferred protection against MAP infection in a goat challenge model.

Vaccination with recombinant Mycobacterium avium subsp. paratuberculosis proteins induces differential immune responses and protects calves against infection by oral challenge

We previously reported the in vitro cellular immune responses to recombinant antigens (rAgs) of Mycobacterium avium subsp. paratuberculosis (MAP). Here we report the differential immune responses and protective efficacy of four rAgs of MAP (85A, 85B, 85C, and superoxide dismutase (SOD)) used with two adjuvants (monophosphoryl lipid A (MPLA) containing synthetic trehalose dicorynomycolate, cell wall skeleton (MPLA) and bovine IL-12), against MAP challenge in calves. Group I was administered the four rAgs with MPLA and IL-12. Group II was administered the four rAgs and MPLA. Group III received MPLA and IL-12, and Group IV MPLA. rAgs induced significant lymphoproliferative responses in vaccinated animals (Groups I and II). All the rAgs induced significant IFN-gamma production from 11 to 23 wk after primary vaccination (APV), except for SOD. Significant increases were noted in CD3(+), CD4(+), CD8(+), CD21(+), CD25(+), and gammadelta(+) cells against all four rAgs in vaccinated animals. rAg-specific expression of IL-2, IL-12p40, IFN-gamma and TNF-alpha was significantly higher in the two vaccinated groups. Culture results found 4/8 animals in Group I, 3/8 animals in Group II, and 3/4 animals in Groups III and IV were positive for MAP in one or more tissues. Among the seven positive animals in Groups I and II, all but one had had <10CFU. Isolation was confined to one tissue in these animals, except in one animal in which MAP was isolated from two tissues. In the control groups (III and IV), MAP was cultured from up to five different tissues with >250CFU. Preliminary data from this study indicates that all four rAgs induced a good Th1 response and conferred protection against MAP infection in calves.

Detection of Human Granulocytic Ehrlichiosis Agent and Borrelia Burgdorferi in Ticks by Polymerase Chain Reaction

Adult ixodid ticks were collected from Westchester County, New York, and Ipswich, Massachusetts, to determine the presence of infection with a human granulocytic ehrlichiosis (HGE) agent by using the polymerase chain reaction (PCR). The presence of Borrelia burgdorferi in ticks collected from New York was also determined by PCR. Of the 229 ticks from New York and 47 ticks from Massachusetts, 9% (22/229) and 25% (12/47) of ticks contained HGE agent, respectively. Fifty-four percent (123/229) of the ticks collected from New York were B. burgdorferi positive; 4% (9/229) of these ticks contained both HGE agent and B. burgdorferi. This finding indicates that animals with Lyme borreliosis may be also exposed to the etiologic agent of HGE. More extensive laboratory diagnosis may be necessary when multiple tick-borne diseases are suspected in animals.

In vitro cellular immune responses to recombinant antigens of Mycobacterium avium subsp. paratuberculosis

ABSTRACT Five recombinant antigens (Ags; 85A, 85B, 85C, superoxide dismutase [SOD], and 35-kDa protein) were purified from Mycobacterium avium subsp. paratuberculosis and evaluated for their ability to stimulate peripheral blood mononuclear cells (PMBCs) from fecal-culture-positive cows (low and medium shedders) and culture-negative healthy cows. Recombinant Ags 85A, 85B, and 85C induced significant lymphocyte proliferation as well as the production of gamma interferon (IFN-γ), interleukin-2 (IL-2), IL-12, and tumor necrosis factor alpha (TNF-α), but not IL-4, from low and medium shedders. The 85 antigen complex did not stimulate PMBC proliferation from culture-negative healthy cows. The 35-kDa protein also induced significant lymphocyte proliferation as well as the production of IFN-γ and IL-4 from low and medium shedders. CD4+ T cells and CD25+ (IL-2R) T cells were stimulated the most by 85A and 85B, while the 35-kDa protein primarily stimulated CD21+ B cells involved in humoral immune responses. Interestingly, SOD was less immunostimulatory than other antigens but strongly induced γδ+ T cells, which are thought to be important in the early stages of infection, such as pathogen entry. These data provide important insight into how improved vaccines against mycobacterial infections might be constructed.

Novel Attenuated Salmonella enterica Serovar Choleraesuis Strains as Live Vaccine Candidates Generated by Signature-Tagged Mutagenesis

ABSTRACT Salmonella enterica serovar Choleraesuis is a host-adapted pathogen that causes swine paratyphoid. Signature-tagged mutagenesis (STM) was used to understand the pathogenicity of S. enterica serovar Choleraesuis in its natural host and also to develop novel attenuated live vaccine candidates against this disease. A library of 960 signature-tagged mutants of S. enterica serovar Choleraesuis was constructed and screened for attenuation in pigs. Thirty-three mutants were identified by the STM screening, and these mutants were further screened for attenuation by in vivo and in vitro competitive growth. Of these, 20 mutants targeting the outer membrane, type III secretion, transporter, lipopolysaccharide biosynthesis, and other unknown proteins were confirmed for attenuation. Five highly attenuated mutants (SC2D2 [ssaV], SC4A9 [gifsy-1], SC6F9 [dgoT], SC12B12 [ssaJ], and SC10B1[spiA]) were selected and evaluated for safety and protective efficacy in pigs by comparison with a commercially available vaccine strain. STM-attenuated live vaccine strains SC4A9 (gifsy-1) and SC2D2 (ssaV) were superior to commercially available live vaccine because they provided both safety and a protective immune response against challenge in pigs.

Epidemiologic Relationship between Fluoroquinolone-Resistant Salmonella enterica Serovar Choleraesuis Strains Isolated from Humans and Pigs in Taiwan (1997 to 2002)

ABSTRACT The emergence of ciprofloxacin-resistant Salmonella enterica serovar Choleraesuis in recent years has become an important public health issue in Taiwan. The resistant strains that cause human infections are considered to be from pigs. In this study, we characterized 157 swine and 42 human Salmonella serovar Choleraesuis isolates by pulsed-field gel electrophoresis (PFGE) and drug susceptibility testing to investigate the epidemiologic relationship among the isolates. By PFGE analyses, two major clusters (clusters GA and GB) were identified. Isolates in cluster GA were of both human and swine origins, while those in cluster GB were from pigs only. Among the various genotypes identified, genotype gt-1a was the most prevalent, which was found in 71% (30 of 42) and 48% (76 of 157) of human and swine isolates, respectively. The susceptibility tests for the 106 gt-1a isolates identified 44 susceptibility profiles and showed that 73% of human isolates and 34% of swine isolates were resistant to three fluoroquinolones (ciprofloxacin, enrofloxacin, and norfloxacin). Our findings indicate that a clonal group of Salmonella serovar Choleraesuis may have been circulating in human and swine populations in Taiwan for years and that the fluoroquinolone-resistant Salmonella serovar Choleraesuis strains most likely evolved from a gt-1a clone that emerged in 2000 and that then caused widespread infections in humans and pigs. Nevertheless, it is still debatable whether those Salmonella infections in humans are caused by isolates derived from pigs, on the basis of the higher fluoroquinolone and other antimicrobial resistance percentages in human isolates than in pig isolates.

Antimicrobial susceptibility of Riemerella anatipestifer isolated from ducks and the efficacy of ceftiofur treatment

The in vitro susceptibilities of 50 field isolates of Riemerella anatipestifer from ducks to ceftiofur and 16 other commonly used antimicrobials were determined. The MIC90 values (MIC refers to minimum inhibitory concentrations) for the antimicrobials used in this study are as follows: penicillin was 16 μg/ml; ceftiofur was 32 μg/ml; cephalothin, chloramphenicol, flumequine, and kanamycin were 64 μg/ml; nalidixic acid, nitrofurantoin, and sulfamethoxazole were 128 μg/ml; amikacin, ampicillin, gentamicin, lincomycin, spec-tinomycin, streptomycin, tetracycline, and trimethoprim were ≥256 μg/ml. The therapeutic efficacy of ceftiofur against a highly lethal experimental R. anatipestifer infection in ducks was also evaluated. All experimental ducks were infected through the infraorbital sinus with 1 ml of 9 × 109 CFU of R. anatipestifer. Ceftiofur (0, 0.25, 0.5, 1, and 2 mg/kg) was injected subcutaneously 5 hours after infection. A single dose of 2 mg/kg resulted in 73% survival as compared with 10% survival in the infected, but untreated controls.