Antimicrobial resistance in bacteria associated with porcine respiratory disease in Australia

The porcine respiratory disease complex greatly affects the health and production of pigs. While antimicrobial agents are used to treat the respiratory infections caused by bacterial pathogens, there is no current information on antimicrobial resistance in Australian pig respiratory bacterial isolates. The aim of this study was to determine the antimicrobial resistance profiles, by determining the minimum inhibitory concentration of nine antimicrobial agents for 71 Actinobacillus pleuropneumoniae, 51 Pasteurella multocida and 18 Bordetella bronchiseptica cultured from Australian pigs. The majority of A. pleuropneumoniae isolates were resistant to erythromycin (89%) and tetracycline (75%). Resistance to ampicillin (8.5%), penicillin (8.5%) and tilmicosin (25%) was also identified. The P. multocida isolates exhibited resistance to co-trimoxazole (2%), florfenicol (2%), ampicillin (4%), penicillin (4%), erythromycin (14%) and tetracycline (28%). While all the B. bronchiseptica isolates showed resistance to beta-lactams (ampicillin, ceftiofur and penicillin), some were resistant to erythromycin (94%), florfenicol (6%), tilmicosin (22%) and tetracycline (39%). The incidence of multiple drug resistance (MDR) varied across the species - in B. bronchiseptica, 27.8% of resistant isolates showed MDR, while 9.1% of the resistant isolates in A. pleuropneumoniae, and 4.8% in P. multocida showed MDR. This study illustrated that Australian pig strains of bacterial respiratory pathogens exhibited low levels of resistance to antimicrobial agents commonly used in the pig industry

Use of a proposed antimicrobial susceptibility testing method for Haemophilus parasuis

The aim of this study was to examine the antimicrobial susceptibility of 97 Haemophilus parasuis cultured from Australian pigs. As there is no existing standard antimicrobial susceptibility technique available for H. parasuis, methods utilising the supplemented media, BA/SN for disc diffusion and test medium broth (TMB) for a microdilution technique, were initially evaluated with the reference strains recommended by the Clinical and Laboratory Standards Institute. The results of the media evaluation suggested that BA/SN and TMB can be used as suitable media for susceptibility testing of H. parasuis. The proposed microdilution technique was then used with 97 H. parasuis isolates and nine antimicrobial agents. The study found that Australian isolates showed elevated minimum inhibitory concentrations (MICs) for ampicillin (1%), penicillin (2%), erythromycin (7%), tulathromycin (9%), tilmicosin (22%), tetracycline (31%) and trimethoprim-sulfamethoxazole (40%). This study has described potential antimicrobial susceptibility methods for H. parasuis and has detected a low percentage of Australian H. parasuis isolates with elevated antimicrobial MICs

Use of a proposed antimicrobial susceptibility testing method for Haemophilus parasuis

The aim of this study was to examine the antimicrobial susceptibility of 97 Haemophilus parasuis cultured from Australian pigs. As there is no existing standard antimicrobial susceptibility technique available for H. parasuis, methods utilising the supplemented media, BA/SN for disc diffusion and test medium broth (TMB) for a microdilution technique, were initially evaluated with the reference strains recommended by the Clinical and Laboratory Standards Institute. The results of the media evaluation suggested that BA/SN and TMB can be used as suitable media for susceptibility testing of H. parasuis. The proposed microdilution technique was then used with 97 H. parasuis isolates and nine antimicrobial agents. The study found that Australian isolates showed elevated minimum inhibitory concentrations (MICs) for ampicillin (1%), penicillin (2%), erythromycin (7%), tulathromycin (9%), tilmicosin (22%), tetracycline (31%) and trimethoprim-sulfamethoxazole (40%). This study has described potential antimicrobial susceptibility methods for H. parasuis and has detected a low percentage of Australian H. parasuis isolates with elevated antimicrobial MICs

Reaction of mouse post-immune sera against <i>Cryptosporidium</i> antigens and the effect of gp900 domain 3 immunization on parasite shedding after challenge.

(A) Mice were immunized 3 times with C. hominis TU502 (n = 5) or zoonotic C. p. parvum (n = 5) lysates intraperitoneally (IP), or once with zoonotic C. p. parvum oocysts (n = 5) orally (PO). Four unimmunized mice served as controls. C. p. parvum lysate immune sera had high IgG level against C. p. parvum and gp900 D3 and lower anti-C. hominis IgG level (Kruskal-Wallis with Dunn’s multiple comparisons test: anti- C. p. parvum vs. anti-gp900 domain 3, p = >0.9999; anti- C. p. parvum IgG vs. anti-C. hominis, p = 0.0267; and anti-gp900 domain 3 vs. anti-C. hominis, p = 0.0216). (B) 3–4-week-old BALB/c mice (n = 5) were immunized with recombinant gp900 D3 protein or PBS (controls, n = 5) on day 0, 35 and 56. Cryptosporidium specific antibodies were measured three weeks after the last immunization. Immunized mice had elevated level of anti-C. p. parvum and anti-gp900 D3 specific IgG compared to controls (Mann Whitney test, p = 0.008 and 0.0079, respectively), while anti-C. hominis IgG was undetectable. (C) gp900 D3 immunized mice had higher anti- C. p. parvum IgA in feces and saliva when compared to unimmunized controls (Mann Whitney test, p = 0.0079 and 0.0097, respectively). (D) gp900 domain 3 immunized and unimmunized control mice were challenged with 106 C. p. parvum oocysts. A quantitative PCR showed a minor difference where oocyst excretion in the systemically D3 immunized mice was delayed by 24 hours as compared with the control. Error bars show SD. (E) Sections of terminal ilea of mice: left image (uninfected mouse) shows a normal villi coated with normal enterocytes; the right image (mouse with cryptosporidiosis) shows the moderately infected enterocytes embedded in the microvillus border (arrows). The infected section is covered by normal healthy layer of enterocytes. (H & E; M– 400x). Scale indicates 20μm.</p

<i>Cryptosporidium</i>-specific IgG in gnotobiotic post-immune sera and <i>C</i>. <i>hominis</i> infection in piglet gut.

3–5-day old gnotobiotic piglets received a primary infection with C. p. anthroponosum TU114 (n = 4) or C. hominis TU502 (n = 8) or remained uninfected (unprimed) and were challenged (secondary infection) with C. p. anthroponosum TU114 (n = 3) or C. hominis TU502 (n = 4) after recovery (~33–45 days after birth). (A) Cryptosporidium-specific IgG was determined from sera collected 10 days post-challenge. Some sera from primed and challenged piglets had elevated anti- C. p. parvum or C. hominis in contrast to undetectable anti-gp900 D3 response in all sera regardless of infections received (unprimed/primed). Each data point is the mean OD 450nm of duplicates at 1:1600 serum dilution obtained when tested against Cryptosporidium antigens. (B) Sections of terminal ilea of gnotobiotic piglets. The left image shows a normal villi coated with normal enterocytes. The right image shows the heavily infected enterocytes with C. hominis forms (arrows) embedded in the microvillus border. The tip of the infected section is covered by irregular and damaged layer of enterocytes (H & E; M– 400x). Scale indicates 20μm.</p

Immune responses against <i>Cryptosporidium</i>-specific antigens in sera of gnotobiotic piglets orally infected with anthroponotic <i>Cryptosporidium</i> spp. oocysts.

Immune responses against Cryptosporidium-specific antigens in sera of gnotobiotic piglets orally infected with anthroponotic Cryptosporidium spp. oocysts.</p

<i>Cryptosporidium</i>-specific IgG in post immune rat sera and the effect of these sera in <i>C</i>. <i>parvum</i> infection in vitro.

Rats were immunized 3 times on day 0, 35 and 56 with sporozoite lysates of C. hominis TU502 (n = 2), zoonotic C. parvum (n = 2) or C. p. anthroponosum TU114 (n = 1), with each immunization containing lysate of 106 excysted oocysts. Additional rats (n = 2) were immunized with or C. p. parvum derived gp900 D3 or PBS as control (n = 1). (A) shows the high level of Cryptosporidium-specific IgG (anti-C. hominis, anti-C. p. parvum IOWA and anti-gp900 D3) in post-immune sera in comparison to serum from the control rat. Values indicate optical densities (OD) measured at 450-nm absorbance of the average of technical duplicates at 1:1600 serum dilution. Post immune C. hominis, C. p. anthroponosum TU114 and control had low level of gp900 domain 3 IgG. (B) The effect of post-immune sera to inhibit intracellular C. parvum infection in vitro (using MDBK cells) was determined by adding sera at 1:100, 1:200, 1:400 and 1:800 dilutions. Infection control was a C. p. parvum IOWA cell culture maintained in growth medium alone. Rate of infection (%) in the presence of sera were normalized using data from infection control. Comparison of infection rate between cultures with post immune and age-matched PBS control was analyzed using 2-way ANOVA with Dunnett’s multiple comparisons test. Data shown are mean ±SD from duplicate assays.</p

<i>Cryptosporidium</i>-specific IgG in serum of <i>C</i>. <i>hominis</i> TU502 immunized alpaca.

Each data point is the mean OD 450nm of duplicates of specified serum dilutions obtained when tested against Cryptosporidium antigens (C. hominis, C. p. parvum IOWA and gp900 D3). An alpaca was immunized with C. hominis TU502 oocyst lysates subcutaneously with CpG and Alhydrogel adjuvants, 7 times at 2–4 weeks intervals. Two weeks after the last immunization serum was collected and the anti-C. hominis TU502, C. p. parvum IOWA and gp900 D3 IgG was determined by ELISA. Statistical comparisons: anti-C. hominis TU502 vs. anti-gp900-d3, p = 0.0021; anti-zoonotic C. p. parvum IOWA vs. anti-gp900-d3, p = 0.0008, Kruskal-Wallis test with Dunn’s multiple comparison test.</p

Scalable cryopreservation of infectious Cryptosporidium hominis oocysts by vitrification.

Cryptosporidium hominis is a serious cause of childhood diarrhea in developing countries. The development of therapeutics is impeded by major technical roadblocks including lack of cryopreservation and simple culturing methods. This impacts the availability of optimized/standardized singular sources of infectious parasite oocysts for research and human challenge studies. The human C. hominis TU502 isolate is currently propagated in gnotobiotic piglets in only one laboratory, which limits access to oocysts. Streamlined cryopreservation could enable creation of a biobank to serve as an oocyst source for research and distribution to other investigators requiring C. hominis. Here, we report cryopreservation of C. hominis TU502 oocysts by vitrification using specially designed specimen containers scaled to 100 μL volume. Thawed oocysts exhibit ~70% viability with robust excystation and 100% infection rate in gnotobiotic piglets. The availability of optimized/standardized sources of oocysts may streamline drug and vaccine evaluation by enabling wider access to biological specimens

Rat immunization regimen and their serum antibody response against <i>Cryptosporidium</i> antigens determined by ELISA^a.

Rat immunization regimen and their serum antibody response against Cryptosporidium antigens determined by ELISAa.</p