A zoospore inhibition technique to evaluate the activity of antifungal compounds against Batrachochytrium dendrobatidis and unsuccessful treatment of experimentally infected green tree frogs (Litoria caerulea) by fluconazole and benzalkonium chloride

Effective and safe treatments of chytridiomycosis in amphibians, caused by Batrachochytrium dendrobatidis, are needed to help prevent mortality in captive programs for threatened species, to reduce the risk of spread, and to better manage the disease in threatened populations. We describe a simple method to determine minimum inhibitory concentrations (MICs) of antifungal agents that involves adding zoospores to various drug concentrations in 96 well plates and microscopic observation after four days. We report results from testing 10 commercially available antifungal compounds: benzalkonium chloride (<0.78 μg/ml), povidone iodine (312.5 μg/ml), amphotericin B (3.125 μg/ml), fluconazole (<1.56 μg/ml), itraconazole (<1.56 μg/ml), enilconazole (<1.56 μg/ml), mercurochrome (6.25 μg/ml), sodium chloride (12.5 mg/ml), methylene blue (<1.56 μg/ml) and Virkon (3.125 μg/ml). For treatment trials of juvenile Litoria caerulea, baths of benzalkonium chloride at 1 mg/L and fluconazole at 25 mg/L were used on 18 experimentally infected frogs per treatment. Although these treatments resulted in longer survival times (mean 43.7 ± 11.3 days) than in the untreated controls (37.9 ± 9.3 days), the mortality rate was still 100%. Higher doses of fluconazole are suggested for further animal trials.     

Evaluation of the in vitro growth-inhibitory effect of epoxomicin on Babesia parasites

Epoxomicin potently and irreversibly inhibits the catalytic activity of proteasomal subunits. Treatment of proliferating cells with epoxomicin results in cell death through accumulation of ubiquinated proteins. Thus, epoxomicin has been proposed as a potential anti-cancer drug. In the present study, the inhibitory effects of epoxomicin on the in vitro growth of bovine and equine Babesia parasites were evaluated. The inhibitory effect of epoxomicin on the in vivo growth of Babesia microti was also assessed. The in vitro growth of five Babesia species that were tested was significantly inhibited (P < 0.05) by nanomolar concentrations of epoxomicin (IC₅₀ values = 21.4 ± 0.2, 4 ± 0.1, 39.5 ± 0.1, 9.7 ± 0.3, and 21.1 ± 0.1 nM for Babesia bovis, Babesia bigemina, Babesia ovata, Babesia caballi, and Babesia equi, respectively). Epoxomicin IC₅₀ values for Babesia parasites were low when compared with diminazene aceturate and tetracycline hydrochloride. Combinations of epoxomicin with diminazene aceturate synergistically potentiated its inhibitory effects in vitro on B. bovis, B. bigemina, and B. caballi. In B. microti-infected mice, epoxomicin caused significant (P < 0.05) inhibition of the growth of B. microti at the non-toxic doses of 0.05 and 0.5 mg/kg BW relative to control groups. Therefore, epoxomicin might be used for treatment of babesiosis.     

Comparison of in vivo and in vitro survival and fecundity rates of the poultry red mite, Dermanyssus gallinae

To assist in the testing of possible antigens in developing a vaccine against the poultry red mite (Dermanyssus gallinae De Geer), a rapid and reliable in vitro screening method is critical. This short paper describes how D. gallinae survival and fecundity rates in an in vivo feeding device compared to that of mites fed using an in vitro method. Results showed that survival of fed D. gallinae females and mites overall was greater in vitro, although there was no difference between male survival and fecundity between in vivo and in vitro designs. The in vitro feeding device described therefore has the potential to provide reliable results, comparable to those obtained by in vivo testing, to allow for the rapid screening of D. gallinae antigens.     

Bacterial flora on Cascades frogs in the Klamath mountains of California

Amphibians are experiencing global declines due in part to the infectious disease chytridiomycosis. Some symbiotic bacteria residents on frog skin have been shown to inhibit the growth of Batrachochytrium dendrobatitis (Bd) but few studies have attempted to fully describe the resident bacterial flora of frog skin. We cultured and sequenced 130 bacterial isolates from frogs collected from the California Klamath Range, recovering predominantly Gram-negative bacteria from 20 higher order taxa and 31 genera. There were also a large number of unclassifiable isolates. Forty-three isolates were assessed for their ability to inhibit the growth of Bd in vitro; of these, two had strong and three had slight anti-Bd activity. We suggest that many bacterial species may play a secondary role in Bd resistance, acting synergistically with inhibitory species. Future research is required in order to characterize these interactions. Understanding the relationships between bacterial strains may be important in predicting and managing the effects of future anti-Bd treatments such as antimicrobial compounds or probiotic bacteria.     

In vitro effects of Musa x paradisiaca extracts on four developmental stages of Haemonchus contortus

This study was carried out to evaluate the in vitro effect of Musa x paradisiaca stem and leaf against the parasitic nematode of small ruminants Haemonchus contortus. Three extracts (aqueous, methanolic and/or dichloromethane) of Musa x paradisiaca stem and leaf were tested in vitro on four developmental stages of H. contortus using egg hatch assay (EHA), larval development assay (LDA), L3 migration inhibition assay (LMI) and adult worm motility assay (AWM). The highly significant (P < 0.0001) ability to stop larval development (inhibition >67% for each extract) and the negative effect of the dichloromethane extract of leaf on adult worm motility (43% of inhibition of motility after 24 h of incubation) compared to the negative controls, suggest anthelmintic properties of Musa x paradisiaca stem and leaf against H. contortus. The active principles responsible for the activity could be secondary metabolites such as terpenoid and flavonoid compounds present in the leaf and stem of the plant.     

Characterization of the in vitro core surface proteome of Mycoplasma mycoides subsp. mycoides, the causative agent of contagious bovine pleuropneumonia

Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides (Mmm) is a severe cattle disease, present in many countries in sub-Saharan Africa. The development of improved diagnostic tests and vaccines for CBPP control remains a research priority. Polyacrylamide gel electrophoresis and mass spectrometry were used to characterize the Triton X-114 soluble proteome of nine Mmm strains isolated from Europe or Africa. Of a total of 250 proteins detected, 67 were present in all strains investigated. Of these, 44 were predicted to be lipoproteins or cytoplasmic membrane-associated proteins and are thus likely to be members of the core in vitro surface membrane-associated proteome of Mmm. Moreover, the presence of all identified proteins in other ruminant Mycoplasma pathogens were investigated. Two proteins of the core proteome were identified only in other cattle pathogens of the genus Mycoplasma pointing towards a role in host–pathogen interactions. The data generated will facilitate the identification and prioritization of candidate Mycoplasma antigens for improved control measures, as it is likely that surface-exposed membrane proteins will include those that are involved in host–pathogen interactions.     

Assessing the efficacy of Duddingtonia flagrans chlamydospores per gram of faeces to control Haemonchus contortus larvae

The aims were (a) to quantify the number of Duddingtonia flagrans chlamydospores per gram of faeces (CPG) recovered from sheep administered with different oral doses and, (b) to describe the relationship between CPG and eggs per gram of faeces (EPG) on the efficacy to reduce Haemonchus contortus infective larvae. Three doses of chlamydospores per kg BW were orally administered during seven days: (T1) non treated control group, (T2) 1 × 10⁶, (T3) 2.5 × 10⁶ and (T4) 5 × 10⁶. Three lambs, infected with H. contortus, were used per group. Faeces were obtained from the rectum of each lamb during the fungal administration period (days 0–6) and for six days after that period. Four coproculture replicates were made from each animal in days 2, 4, 6, 8 and 10. A higher chlamydospore dose produced higher CPG in faeces (p < 0.05), but a clear dose dependent effect was not found either in the larvae reduction or in the CPG:EPG ratio. When ratios were re-analyzed, independently of the treatment groups of origin, a better efficacy was obtained with a ratio from 5 to 10 CPG:EPG and a higher ratio (>10 per egg) showed a lower reduction efficacy (p < 0.05). The binomial analysis showed that for each unit of increment in CPG:EPG ratio there was a reduction of larvae number until a point (between 5 and 10 CPG:EPG) where no further reduction was detected. The surface response test indicated that the number of larvae was reduced by CPG until possible saturation. The highest CPG:EPG ratios did not necessarily improve efficacy of D. flagrans.     

Attenuation of virulence of Lawsonia intracellularis after in vitro passages and its effects on the experimental reproduction of porcine proliferative enteropathy

Non-pathogenic Lawsonia intracellularis variants have been obtained through multiple passages in cell culture but there is no information regarding the number of passages necessary to attenuate a pathogenic isolate. The present study evaluated the susceptibility of pigs to L. intracellularis after 10, 20 and 40 passages in vitro. Three groups (six animals/group) were inoculated with pure culture of L. intracellularis on passage 10, 20 or 40 and one group with placebo. The animals were monitored for clinical signs, fecal shedding and serological IgG response during 28 days post-inoculation. Gross and histologic lesions and the level of infection based on the amount of L. intracellularis-specific antigen in the intestinal mucosa identified by immunohistochemistry were evaluated in two animals from each group on days 14, 21 and 28. Animals inoculated with passages 10 and 20 demonstrated proliferative lesions typical of porcine proliferative enteropathy associated with the presence of Lawsonia-specific antigen in the intestinal mucosa. Passage 40-inoculated pigs did not show proliferative lesions or presence of Lawsonia antigen at any time point throughout the study. Similar patterns of the fecal shedding were observed in passage 10 and 20-infected pigs but those infected with passage 40 shed for a short period. Serological IgG responses in passage 10 and 20-inoculated pigs were detected from day 14 post-infection but not at all in passage 40-inoculated animals. These results demonstrate attenuation of the virulence properties of L. intracellularis between 20 and 40 cell passages in vitro. This information will be valuable for design of future experimental models and for studying the mechanisms involved in the attenuation of L. intracellularis virulence.     

Effects of the acid-tolerant engineered bacterial strain Megasphaera elsdenii H6F32 on ruminal pH and the lactic acid concentration of simulated rumen acidosis in vitro

The aim of this study was to examine the effects of the acid-tolerant engineered bacterial strain Megasphaera elsdenii H6F32 (M. elsdenii H6F32) on ruminal pH and the lactic acid concentrations in simulated rumen acidosis conditions in vitro. A mixed culture of ruminal bacteria, buffer, and primarily degradable substrates was inoculated with equal numbers of M. elsdenii H6 or M. elsdenii H6F32. The pH and lactic acid concentrations in the mixed culture were determined at 0, 2, 4, 6, 8, 10, 12, 14, 16, and 18 h of incubation. Acid-tolerant M. elsdenii H6F32 reduced the accumulation of lactic acid and increased the pH value. These results indicate that acid-tolerant M. elsdenii H6F32 could be a potential candidate for preventing rumen acidosis.     

Salmonella Antimicrobial Activity of Selected Strains of Enterolactobacillus Species Isolated from the Gastrointestinal Tract of the Horse

The gastric mucosa and the mucosa of the right and left dorsal colon were biopsied in each of the 15 horses, and a total of 45 samples were collected. Mucosal samples were cultured using a Lactobacillus enrichment broth. While numerous Lactobacillus strains were identified, Lactobacillus reuteri was the most common organism identified. Sixteen strains of Lactobacillus reuteri were selected for antimicrobial testing. Salmonella antimicrobial activity was identified in six out of 16 strains tested. Organisms with Salmonella antimicrobial activity were cultured from the stratified squamous epithelium of the stomach and the mucosa of the right and left dorsal colon.     

Molecular detection of Theileria and Babesia infections in cattle

This study was carried out to determine the presence and distribution of tick-borne haemoprotozoan parasites (Theileria and Babesia) in apparently healthy cattle in the East Black Sea Region of Turkey. A total of 389 blood samples were collected from the animals of various ages in six provinces in the region. Prevalence of infection was determined by reverse line blot (RLB) assay. The hypervariable V4 region of the 18S ribosomal RNA (rRNA) gene was amplified with a set of primers for members of the genera Theileria and Babesia. Amplified PCR products were hybridized onto a membrane to which generic- and species-specific oligonucleotide probes were covalently linked. RLB hybridization identified infection in 16.19% of the samples. Blood smears were also examined microscopically for Theileria and/or Babesia spp. and 5.14% were positive. All samples shown to be positive by microscopy also tested positive with RLB assay. Two Theileria (T. annulata and T. buffeli/orientalis) and three Babesia (B. bigemina, B. major and Babesia sp.) species or genotypes were identified in the region. Babesia sp. genotype shared 99% similarity with the previously reported sequences of Babesia sp. Kashi 1, Babesia sp. Kashi 2 and Babesia sp. Kayseri 1. The most frequently found species was T. buffeli/orientalis, present in 11.56% of the samples. T. annulata was identified in five samples (1.28%). Babesia infections were less frequently detected: B. bigemina was found in three samples (0.77%), B. major in two samples (0.51%) and Babesia sp. in five samples (1.28%). A single animal infected with T. buffeli/orientalis was also infected with B. bigemina.     

Prevalence and antibiotic resistance profiles of Enterococcus species in chicken at slaughter level; absence of vanA and vanB genes in E. faecalis and E. faecium

The prevalence of enterococci in neck skin samples of poultry from Ankara region in Turkey was investigated and their antibiotic resistance patterns were determined. In the study, 83 of 106 analyzed neck skin samples were positive for Enterococcus, with E. faecium as the most prevalent species (48%) followed by E. durans (23%) and E. faecalis (19%). Lower numbers were detected for E. gallinarum, E. hirae, E. mundtii and E. casseliflavus. Using the disc diffusion method, it was established that over 90% of E. faecium and E. faecalis isolates were high-level resistant against erythromycin and tetracycline. Four E. faecium isolates were additionally resistant to chloramphenicol, gentamicin and streptomycin, though they were susceptible to penicillin G. The most frequently observed multiple resistance in E. faecium (25%) was against erythromycin, tetracycline, chloramphenicol and streptomycin. Of the E. faecalis isolates, 44% were multiple resistant to erythromycin, tetracycline and streptomycin. Vancomycin resistance could not be demonstrated phenotypically and vanA or vanB genes were not detected by multiplex PCR in any of the isolates. Nevertheless, the observed resistance patterns are of concern for public health.     

Antimicrobial susceptibility testing of Spanish field isolates of Brachyspira hyodysenteriae

This study is the first conducted in Spain to evaluate antimicrobial susceptibility of field isolates of Brachyspira hyodysenteriae. One hundred and eight isolates of the bacterium, recovered from different Spanish swine farms between 2000 and 2007, were investigated. The minimum inhibitory concentrations (MIC) of erythromycin, tylosin, tiamulin, valnemulin, clindamycin and lincomycin were determined using a broth microdilution technique. Most of the isolates showed poor susceptibility to erythromycin (MIC₉₀ > 256 μg/ml), tylosin (MIC₉₀ > 256 μg/ml), clindamycin (MIC₉₀ > 4 μg/ml) and lincomycin (MIC₉₀ = 128 μg/ml). Reduced susceptibility to tiamulin and valnemulin was observed with a MIC > 2 μg/ml in 17.6% and 7.41% of the B. hyodysenteriae isolates, respectively. Moreover, a survival analysis permitted the detection of an increasing trend in the MIC values for almost all the antimicrobials used in the treatment of swine dysentery when comparing recent isolates (from 2006 to 2007) with those recovered in earlier years (between 2000 and 2004).     

Factors affecting seroprevalence of Toxoplasma gondii in the endangered Iberian lynx (Lynx pardinus)

Wild felids are considered important in maintaining the sylvatic cycle of Toxoplasma gondii. Although, T. gondii antibodies have been reported in several species of wild felids, little is known of the epidemiology and risk factors associated with T. gondii infection in wild cats. The Iberian lynx (Lynx pardinus) is the most endangered felid species in the world. In the present study, seroprevalence and associated risk factors for T. gondii infection in a large population of Iberian lynx in Spain were determined. Serum samples from 129 Iberian lynx collected from 2005 to 2009 and 85 wild rabbits (Oryctolagus cuniculus), sharing the habitat with the Iberian lynx, were tested for antibodies to T. gondii by the modified agglutination test (MAT) using a cut-off value of 1:25. Antibodies to T. gondii were found in 81 of 129 (62.8%) Iberian lynx. Seroprevalence to T. gondii in Iberian lynx significantly increased with age (P < 0.001). T. gondii seroprevalences were similar in free-ranging (66.7% of 93) and wild-caught captive lynx (69% of 84) but significantly lower in captive-born lynx (22.5% of 40). Seroprevalence was higher in lynx with concurrent Cytauxzoonfelis (88% of 25) but not with concurrent Feline Leukemia Virus (FeLV) infection (53.8% of 13). There were no significant differences in seroprevalence between sexes, geographic region and year of sample collection (2005–2009). Oocysts of T. gondii were not detected microscopically in fecal samples from 58 lynx. Wild rabbits are considered the most important food for the lynx. Antibodies to T. gondii were found in 14 (11.9%) of 85 rabbits tested. The present results indicate that T. gondii infection is widespread in the two areas where Iberian lynx survive in Spain. The fact that four captive-born lynx seroconverted was indication of contact with T. gondii also in the Captive Breeding Centers, hence, control measures to prevent T. gondii infection would be necessary in these centers.     

Gastrointestinal parasites of cavy (Cavia aperea aperea) in southern Brazil

The aim of this study was to evaluate the gastrointestinal parasitism in Cavia aperea aperea (cavy), captured in the central region of Rio Grande do Sul State. Fecal samples from five free-living cavies were collected for research of parasites. Samples were analyzed by the centrifugal-flotation method with zinc sulfate and parasites were identified microscopically based on (oo)cyst and egg size and morphology. Cysts of Giardia sp. and (oo)cysts of Cryptosporidium sp. and Cystoisospora sp. were observed in one or more cavies. Eggs of Paraspidodera uncinata were observed in three of the five rodents. All infected animals showed mild infection by parasite. This is the first report of Giardia sp., Cryptosporidium sp. and Cystoisospora sp. in Cavia a. aperea.     

Diversity of Campylobacter jejuni and Campylobacter coli Genotypes from Human and Animal Sources from Rio de Janeiro, Brazil

To compare the genotypes of Campylobacter jejuni and Campylobacter coli isolates of human and animal origin collected in Rio de Janeiro City, 30 C. jejuni and 35 C. coli isolates from animal sources (n = 45) and human patients with gastroenteritis (n = 20) were genotyped by PCR-based techniques, namely random amplified polymorphic DNA (RAPD-PCR) and enterobacterial repetitive intergenic consensus sequence (ERIC-PCR). RAPD-PCR identified 50 types and ERIC-PCR identified 22 genotypes, among the 65 Campylobacter isolates. Both PCR methods discriminated the C. jejuni and C. coli groups of isolates. Combining the results of both methods, no single genotype was shared between isolates from human and animal sources. Two groups of two C. coli isolates each with identical genotypes were found among poultry and pig isolates. A high level of genetic diversity observed among the Campylobacter isolates suggests lack of overlap between isolates from different sources.     

Occurrence of Mycobacterium avium subspecies paratuberculosis and Neospora caninum in Alberta cow-calf operations

Mycobacterium avium subsp. paratuberculosis (MAP) and Neospora caninum (NC) are two pathogens causing important production limiting diseases in the cattle industry. Significant impacts of MAP and NC have been reported on dairy cattle herds, but little is known about the importance, risk factors and transmission patterns in western Canadian cow-calf herds. In this cross-sectional study, the prevalence of MAP and NC infection in southwest Alberta cow-calf herds was estimated, risk factors for NC were identified, and the reproductive impacts of the two pathogens were assessed.