Zoonotic Isolates Research Articles

Detection and evaluation of the antimicrobial activity of Micrococcin P1 isolated from commensal and environmental staphylococcal isolates against MRSA

Bacteriocins (of different origins) have been proposed as promising alternatives to face antimicrobial resistance-associated health problems. Isolates of the Staphylococcus genus are well-known bacteriocin producers, especially coagulase-negative species. Twenty-eight bacteriocin-producing staphylococcal isolates were selected from a previous study for in-depth characterisation. The antimicrobial activities (AA) of the producing isolates were studied by the spot-on-lawn method and their crude cell-free supernatants (CFS) and butanol extracts (BT) were evaluated by agar diffusion assays against six indicator bacteria, including multidrug-resistant and zoonotic isolates (such as Listeria monocytogenes or methicillin-resistant Staphylococcus aureus [MRSA]). Six bacteriocin-producing isolates showed AA in their CFS, whereas all staphylococcal BT extracts inhibited at least one of the tested indicator bacteria. Micrococcin P1 (MP1) bacteriocin was detected by mass spectrometry in four producing isolates: Staphylococcus aureus-C5802, Staphylococcus hominis-C5835, Staphylococcus sciuri-X3041, and -X3011. Growth curves performed with CFS and BT extracts of the four MP1 producers revealed a strong AA profile against MRSA and Listeria monocytogenes, even when considerably diluted. Moreover, synergism between the BT extract of MP1-producing Staphylococcus sciuri-X3041 and several antibiotics against an MRSA indicator was observed: BT-clindamycin (> 80%) and BT-oxacillin (30%) combinations. For the BT-chloramphenicol combination, synergism and near synergism values were observed in 37% of the combinations. Competition studies revealed potent inhibitory effects of the MP1-producing isolates against the MRSA indicator. These results help to identify Staphylococcus isolates or their bacteriocins as interesting candidates for potential future applications.

Reduced susceptibility to carbapenems in Enterobacteriaceae and antimicrobial resistance profile of Escherichia coli strains isolated from clinical and zoonotic sources in the Bamenda Municipality, Cameroon.

food-producing animals harbour pathogenic and antibiotic resistant bacteria which can be transmitted to humans. Resistance to carbapenems may complicate treatment resulting to debilitating consequences. This study aimed at determining the susceptibility of Enterobacteriaceae to carbapenems and to compare the resistant patterns of E. coli strains isolated from clinical and zoonotic sources. this was a cross-sectional study involving patients presenting at the Bamenda Regional Hospital and abattoir samples. Clinical samples (faeces and urine) and zoonotic samples (cattle faeces) were cultured and isolates identified using API-20E. Enterobacteriaceae isolates were tested for their susceptibility to Carbapenems. The susceptibility of E. coli was tested against eight antibiotics on Mueller Hinton agar. Data was analysed using SPSS version 20. Enterobacteriaceae isolates from clinical specimen showed susceptibility of 93.3% to carbapenems. Out of 208 isolates 14 (6.7%) were Carbapenem-resistant Enterobacteriaceae (CRE) while 30 (14.4%) showed intermediate resistance and 164 (78.9%) were susceptible. The predominant CRE were Proteus (7/16, 43.8%), Providencia (3/15, 20.0%) and E. coli (4/60, 6.7%) with E. coli being the most clinically significant CRE. Multiple drug resistance (MDR) was observed in 83% of E. coli isolates, with the highest resistance being against vancomycin (90, 81.8%), azithromycin (69, 62.7%) and doxycycline (68, 61.8%). Clinical isolates were significantly (P<0.05) more resistant to azithromycin, trimethoprim-suphamethoxazole and gentamicin than zoonotic isolates. CRE were detected among isolates and a high rate of multiple drug resistance was observed among E. coli isolates. Proper antibiotic policies and good hygiene/sanitation measures may curb the development/spread of CRE and MDR E. coli.

Diverse Genotypes and Species of Cryptosporidium in Wild Rodent Species from the West Coast of the USA and Implications for Raw Produce Safety and Microbial Water Quality.

Cryptosporidium spp. are protozoan parasites that infect perhaps all vertebrate animals, with a subset of species and genotypes that function as food- and waterborne pathogens. The objective of this work was to collate the Cryptosporidium species and genotypes from common wild rodents on the west coast of the USA and update the information regarding the zoonotic potential of Cryptosporidium from these ubiquitous wild species. Representative sequences of the 18S rRNA gene for a unique set of Cryptosporidium isolates obtained from deer mice, house mice, mountain beavers, yellow-bellied marmot, long-tailed vole, California ground squirrels, Belding’s ground squirrels, and a golden-mantled ground squirrel in GenBank were selected for phylogenetic analysis. Phylogenetic and BLAST analysis indicated that 4 (18%) of the 22 unique Cryptosporidium sequences from these wild rodent species were 99.75% to 100% identical to known zoonotic species (C. parvum, C. ubiquitum, C. xiaoi), suggesting that a minority of these representative Cryptosporidium isolates could have a public health impact through food and waterborne routes of human exposure. These zoonotic isolates were shed by deer mice and a yellow-bellied marmot from California, and from a mountain beaver trapped in Oregon. In addition, the group of unique Cryptosporidium isolates from deer mice and ground dwelling squirrels exhibited considerable DNA diversity, with multiple isolates appearing to be either host-limited or distributed throughout the various clades within the phylogenetic tree representing the various Cryptosporidium species from host mammals. These results indicate that only a subset of the unique Cryptosporidium genotypes and species obtained from wild rodents on the US west coast are of public health concern; nevertheless, given the geographic ubiquity of many of these host species and often high density at critical locations like municipal watersheds or produce production fields, prudent pest control practices are warranted to minimize the risks of water- and foodborne transmission to humans.

Zoonotic bacteria in the food chain: antimicrobial resistance and associated factors in Portugal

Abstract Antimicrobial resistance (AMR) poses a threat to public health and animal food-production plays a crucial role in AMR spread. We thus aimed to characterize AMR profiles and associated factors in zoonotic bacterial isolates in Portugal. Data from the Portuguese AMR surveillance program on animal populations and derived food, 2014-2018, was used. AMR frequency was described in E coli, Campylobacter and Salmonella isolates from poultry and pigs, according to antibiotic and samples tested. For Salmonella, AMR associated factors were studied: animal populations (broilers, laying hens, pigs) and derived meat, sampling stage (farm, slaughterhouse, processing plant), sample type (environmental, carcase, food), sampler (HACCP, industry, official, official and industry), sample context (control and eradication programmes, monitoring), year (2014-2018) and season. Logistic regression was applied to estimate crude (OR) and adjusted odds ratio (aOR) with 95% confidence intervals (95%CI). The final model was obtained using a backward stepwise method. We assessed 2157 E Coli, 561 Campylobacter and 826 Salmonella isolates. AMR rates were higher to cefepime (91-98%) in E coli in all sample types; cefotaxime (100%) in E coli in broiler's meat; nalidixic acid (79-96%) in E coli and Campylobacter in poultry and derived meat; ciprofloxacin (50-98%) in all bacteria isolates from poultry and derived meat. For Salmonella, AMR was more likely in pig's meat (OR 3.66; 95%CI:1.19-2.82), slaughterhouses (OR 10.18; 95%CI:4.69-26.70), carcases (OR 5.90;95%CI:3.74-9.69). The final model indicated a lesser chance of AMR in laying hens (aOR .21; 95%CI:.11-.37), industry sampling (aOR .25; 95%CI:.07-.73), 2018 (aOR .24; 95%CI:.13-.42), and a higher chance in summer (aOR 2.39; 95%CI:1.34-4.28). The findings support the need for control measures on the use of key antibiotics in veterinary medicine. AMR occurrence in Portugal seems to be related to summer, although less in laying hens and in 2018. Key messages AMR in bacterial isolates suggests the need for integrated actions to mitigate and control the use of 3rd and 4rd generation cephalosporins, fluoroquinolones and quinolones in animal production. AMR occurrence in Portugal seems to be further related to summer conditions, while comparing to other seasons.

Five Complete Genome Sequences Spanning the Dutch Streptococcus suis Serotype 2 and Serotype 9 Populations.

The zoonotic pathogen Streptococcus suis can cause septicemia and meningitis in humans. We report five complete genomes of Streptococcus suis serotype 2 and serotype 9, covering the complete phylogeny of serotype 9 Dutch porcine isolates and zoonotic isolates. The isolates include the model strain S10 and the Dutch emerging zoonotic lineage.

Prominent Binding of Human and Equine Fibrinogen to Streptococcus equi subsp. zooepidemicus Is Mediated by Specific SzM Types and Is a Distinct Phenotype of Zoonotic Isolates.

Streptococcus equi subsp. zooepidemicus is an important pathogen in horses that causes severe diseases such as pneumonia and abortion. Furthermore, it is a zoonotic agent, and contact with horses is a known risk factor. In this study, we investigated the working hypothesis that the zoonotic potential varies among S. equi subsp. zooepidemicus strains in association with differences in M-like protein-mediated binding of host plasma proteins. We demonstrate via in-frame deletion mutagenesis of two different S. equi subsp. zooepidemicus strains that the M-like protein SzM is crucial for the binding of fibrinogen to the bacterial surface and for survival in equine and human blood. S. equi subsp. zooepidemicus isolates of equine and human origins were compared with regard to SzM sequences and binding of equine and human fibrinogens. The N-terminal 216 amino acids of the mature SzM were found to exhibit a high degree of diversity, but the majority of human isolates grouped in three distinct SzM clusters. Plasma protein absorption assays and flow cytometry analysis revealed that pronounced binding of human fibrinogen is a common phenotype of human S. equi subsp. zooepidemicus isolates but much less so in equine S. equi subsp. zooepidemicus isolates. Furthermore, binding of human fibrinogen is associated with specific SzM types. These results suggest that SzM-mediated binding of human fibrinogen is an important virulence mechanism of zoonotic S. equi subsp. zooepidemicus isolates.

Genetic and serologic surveillance of canine (CIV) and equine (EIV) influenza virus in Nuevo León State, México.

BackgroundDespite the uncontrolled distribution of the Influenza A virus through wild birds, the detection of canine influenza virus and equine influenza virus in Mexico was absent until now. Recently, outbreaks of equine and canine influenza have been reported around the world; the virus spreads quickly among animals and there is potential for zoonotic transmission.MethodsAmplification of the Influenza A virus matrix gene from necropsies, nasal and conjunctival swabs from trash service horses and pets/stray dogs was performed through RT-PCR. The seroprevalence was carried out through Sandwich enzyme-linked immunosorbent assay system using the M1 recombinant protein and polyclonal antibodies anti-M1.ResultsThe matrix gene was amplified from 13 (19.11%) nasal swabs, two (2.94%) conjunctival swabs and five (7.35%) lung necropsies, giving a total of 20 (29.41%) positive samples in a pet dog population. A total of six (75%) positive samples of equine nasal swab were amplified. Sequence analysis showed 96–99% identity with sequences of Influenza A virus matrix gene present in H1N1, H1N2 and H3N2 subtypes. The phylogenetic analysis of the sequences revealed higher identity with matrix gene sequences detected from zoonotic isolates of subtype H1N1/2009. The detection of anti-M1 antibodies in stray dogs showed a prevalence of 123 (100%) of the sampled population, whereas in horses, 114 (92.68%) positivity was obtained.ConclusionThe results unveil the prevalence of Influenza A virus in the population of horses and dogs in the state of Nuevo Leon, which could indicate a possible outbreak of equine and Canine Influenza in Mexico. We suggest that the prevalence of Influenza virus in companion animals be monitored to investigate its epizootic and zoonotic potential, in addition to encouraging the regulation of vaccination in these animal species in order to improve their quality of life.

Risk factors of Salmonella infection in laying hens in Menoua Division, Western region of Cameroon (Central Africa)

Salmonella infections in poultry farms are overlooked in many African countries; yet these infections are mostly zoonotic with impact on both poultry industry and public health. Considering the impact of Salmonella in laying hens, and the role of laying hens as a source of Salmonella outbreak in human, knowledge of the status of Salmonella on laying hen farms as well as the factors influencing the presence of Salmonella is important. In a cross sectional study, cloacal swabs were collected from 270 commercial laying hens on 27 farms located in Menoua Division. These samples were cultured on standard media. A questionnaire was used to collect information on animals, farms and farmer’s characteristics. The prevalence of Salmonella was 93.34%; three zoonotic isolates namely S. Enteritidis (75.90%), S. Paratyphi (11.90%), and S. Typhimurium (5.60%) were identified. The location of farms was significantly associated with presence of Salmonella, and the risk of infection was 10-fold higher in Nkong-ni than Santchou (p < 0.05). Other potential risk factors such as flock size, age of the farm (infrastructure), or water source were not associated with Salmonella infection. The prophylactic measures against avian diseases in the country must include measures against Salmonella to protect poultry industry and public health.

Strongyloides stercoralis infection in imported and local dogs in Switzerland: from clinics to molecular genetics.

Strongyloides stercoralis is a worldwide-distributed intestinal nematode affecting mainly humans and dogs. Canine strongyloidosis is generally characterised by diarrhoea, malabsorption and bronchopneumonia, and may be fatal in cases of impaired immunity. In recent years, molecular and epidemiological studies suggested that host-adapted populations of S. stercoralis with different zoonotic potential may exist. Clinical and subclinical cases of S. stercoralis infection have been increasingly diagnosed in imported (France, Belgium, Bulgaria) and locally born dogs in Switzerland, showing that this parasite is currently circulating in Europe. Three of these clinical cases will be described here. All three dogs presented severe disease, characterised by harsh diarrhoea, dehydration, vomiting, respiratory and/or neurologic signs, and needed intensive care and hospitalisation. One of these dogs was related to a Swiss breeding kennel, in which the infection was subsequently diagnosed in several other dogs. Faeces were analysed by three coproscopical methods including (i) the Baermann technique, which consistently identified the typical S. stercoralis first-stage larvae in both clinical and subclinical infections, (ii) the sedimentation-zinc chloride flotation and (iii) sodium acetate-acetic acid-formalin concentration (SAFC) methods, which allowed the additional identification of parasitic females and/or eggs in two of the clinical cases. Interestingly, S. stercoralis isolated from all three independent clinical cases exhibited an identical genetic background on the nuclear 18S rDNA (fragment involving hypervariable regions I and IV) and the mitochondrial cytochrome oxidase subunit I (cox1) loci, similar to that of zoonotic isolates from other geographical regions, and not to that of dog-adapted variants. Due to the clinical relevance and zoonotic potential of this parasite, the awareness of both diagnosticians and clinicians is strongly required.

Molecular detection of Giardia duodenalis and Cryptosporidium spp. in canine faecal samples contaminating public areas in Northern Italy.

Giardia duodenalis and Cryptosporidium spp. are common intestinal pathogens of humans and animals. Dogs may be infected by zoonotic isolates of G. duodenalis and Cryptosporidium spp. that, consequently, have high interest under public health perspective. This study estimated the occurrence of these protozoa in canine faeces polluting public areas of Padua municipality (Northern Italy), towards a potential evaluation of health risks for dogs and humans. A total of 705 canine stools was collected in green (n=270) and urban (n=435) areas and processed by duplex real-time PCR and real-time PCR SYBR® Green I for the detection of both protozoa. Positive samples were submitted to specific nested PCRs (i.e. β-giardin/SSU-rRNA genes for Giardia; SSU-rRNA gene for Cryptosporidium) to obtain detailed information on the isolates retrieved.Giardia and Cryptosporidium prevalence were 28.9% and 1.7%, respectively. Twenty-one Giardia-positive samples were successfully identified as dog-specific assemblages C and D, and 1 as the human-specific assemblage B. One isolate was identified as Cryptosporidium canis, while the other 11 were confirmed to belong to the Cryptosporidium parvum species complex. Contrariwise to the Cryptosporidium low prevalence, the wide distribution of Giardia suggests a high risk of infection for dogs attending public areas. Although data indicate a limited risk for human health, it is necessary to improve general education to reduce canine faecal pollution towards a widespread awareness of health risks.

Streptococcal Adhesin P (SadP) contributes to Streptococcus suis adhesion to the human intestinal epithelium.

BackgroundStreptococcus suis is a zoonotic pathogen, causing meningitis and septicemia. We previously demonstrated that the gastrointestinal tract (GIT) is an entry site for zoonotic S. suis infection. Here we studied the contribution of Streptococcal adhesin Protein (SadP) to host-pathogen interaction at GIT level.MethodsSadP expression in presence of Intestinal Epithelial Cells (IEC) was compared with expression of other virulence factors by measuring transcript levels using quantitative Real Time PCR (qRT-PCR). SadP variants were identified by phylogenetic analysis of complete DNA sequences. The interaction of SadP knockout and complementation mutants with IEC was tested in vitro.ResultsExpression of sadP was significantly increased in presence of IEC. Sequence analysis of 116 invasive strains revealed five SadP sequence variants, correlating with genotype. SadP1, present in zoonotic isolates of clonal complex 1, contributed to binding to both human and porcine IEC and translocation across human IEC. Antibodies against the globotriaosylceramide Gb3/CD77 receptor significantly inhibited adhesion to human IEC.ConclusionSadP is involved in the host-pathogen interaction in the GIT. Differences between SadP variants may determine different affinities to the Gb3/CD77 host-receptor, contributing to variation in adhesion capacity to host IEC and thus to S. suis zoonotic potential.

Dissemination of erm(B) and its associated multidrug-resistance genomic islands in Campylobacter from 2013 to 2015

A total of 1372 Campylobacter isolates (1107 Campylobacter coli and 265 Campylobacter jejuni) were obtained from 3462 samples collected from slaughterhouses and farms in three representative regions of China (Shandong, Guangdong, and Shanghai) over three successive years (2013–2015). Of these, 84 (84/1372, 6.1%) were erm(B)-positive, and all 84 positive isolates were identified as C. coli (83 chicken isolates and one swine isolate). The prevalence of erm(B) in Campylobacter isolates was compared amongst the different regions and between the three years investigated. The rates of erm(B)-positive Campylobacter in Guangdong increased remarkably over the experimental period (3.8% to 22.8%), while their higher rates observed in Shanghai (4.4%) and Shandong (2.4%) occurred in 2015 and 2014. Further, 72 erm(B)-positive isolates were associated with the type V and VI multidrug-resistance genomic islands (MDRGIs), which have previously only been identified in human Campylobacter isolates, while one isolate of chicken origin contained the type II MDRGI, which has previously been detected in swine isolates. Expansion of the erm(B) in Campylobacter with similar PFGE and MLST type from chicken isolates from Shanghai and Guangdong to human isolates identified previously in Shanghai was also observed. The findings in this study confirmed previously observed trend of dissemination of erm(B) and MDRGIs in zoonotic Campylobacter isolates and provide new insights into the prevalence of erm(B)-positive Campylobacter isolates in chickens and swine from three representative regions of China over a consecutive 3-year period.

An emerging zoonotic clone in the Netherlands provides clues to virulence and zoonotic potential of Streptococcus suis.

Streptococcus suis is a zoonotic swine pathogen and a major public health concern in Asia, where it emerged as an important cause of bacterial meningitis in adults. While associated with food-borne transmission in Asia, zoonotic S. suis infections are mainly occupational hazards elsewhere. To identify genomic differences that can explain zoonotic potential, we compared whole genomes of 98 S. suis isolates from human patients and pigs with invasive disease in the Netherlands, and validated our observations with 18 complete and publicly available sequences. Zoonotic isolates have smaller genomes than non-zoonotic isolates, but contain more virulence factors. We identified a zoonotic S. suis clone that diverged from a non-zoonotic clone by means of gene loss, a capsule switch, and acquisition of a two-component signalling system in the late 19th century, when foreign pig breeds were introduced. Our results indicate that zoonotic potential of S. suis results from gene loss, recombination and horizontal gene transfer events.

Characterization of Five Zoonotic Streptococcus suis Strains from Germany, Including One Isolate from a Recent Fatal Case of Streptococcal Toxic Shock-Like Syndrome in a Hunter.

A Streptococcus suis isolate from a German hunter with streptococcal toxic shock-like syndrome (STSLS) and four additional zoonotic isolates were genotyped as mrp(+) epf* (variant 1890) sly(+) cps2(+). All five zoonotic German strains were characterized by high multiplication in human blood samples ex vivo, but induction of only low levels of proinflammatory cytokines compared to a Chinese STSLS strain.

Use of a commercial enzyme-linked immunosorbent assay for rapid detection of Giardia duodenalis in dog stools in the environment

Giardia duodenalis is considered a potentially zoonotic protozoan. Some animal species, including infected dogs, may play an important role in the spread of Giardia cysts through environmental contamination with their feces. In the present study, a commercial enzyme-linked immunosorbent assay (ELISA) was used to examine 143 samples of dog feces collected in urban areas as an indicator of the risk of field contamination. Using a Bayesian statistical approach, the ELISA showed a sensitivity of 88.9% and a specificity of 95.8% with positive and negative predictive values of 89.6% and 95.4%, respectively. The test affords the advantage of rapid processing of fecal samples without a complex technical structure and extensive costly labor. Moreover, the present results show that the assay provides public health veterinarians with a practical tool that can be used in screening programs, as a valid alternative or as an adjunct to other tests, in order to assess the biological risk of exposure to G. duodenalis cysts from dogs in human settlements. However, the test may not be completely accurate for human health risk evaluation, as it does not discriminate between zoonotic and non-zoonotic isolates.

Evolution of the Hepatitis E Virus Polyproline Region: Order from Disorder

The hepatitis E virus (HEV) polyproline region (PPR) is an intrinsically unstructured region (IDR). This relaxed structure allows IDRs, which are implicated in the regulation of transcription and translation, to bind multiple ligands. Originally the nucleotide variability seen in the HEV PPR was assumed to be due to high rates of insertion and deletion. This study shows that the mutation rate is about the same in the PPR as in the rest of the nonstructural polyprotein. The difference between the PPR and the rest of the polyprotein is due to the higher tolerance of the PPR for substitutions at the first and second codon positions. With this higher promiscuity there is a shift in nucleotide occupation of these codons leading to translation of more cytosine residues: a shift that leads to more proline, alanine, serine, and threonine being encoded rather than histidine, phenylalanine, tryptophan, and tyrosine. This pattern of amino acid usage is typical of proline-rich IDRs. Increased usage of cytosine also leads to >22% of all amino acids in the PPR being prolines. Alignments of PPR sequences from HEV strains representing all genotypes indicate that all zoonotic isolates share an ancestor, and the carboxyl half of the PPR is more tolerant of mutations than the amino half. The evolution of HEV PPR, in contrast with that of the rest of the nonstructural polyprotein, is molded by pressures that lead toward increased proline usage with a corresponding decrease in the usage of aromatic amino acids, favoring formation of IDR structures.

Detection and Characterization ofGiardia duodenalisandCryptosporidiumspp. on Swine Farms in Ontario, Canada

As part of the C-EnterNet surveillance program of the Public Health Agency of Canada, 122 pooled swine manure samples from 10 farms in Ontario, Canada were collected and tested for Giardia and Cryptosporidium. Giardia duodenalis cysts and Cryptosporidium spp. oocysts were detected using immunofluorescence microscopy. Nested-polymerase chain reaction protocols were performed to amplify the small subunit rRNA gene and the β-giardin gene for G. duodenalis, and the small subunit rRNA gene and the heat shock protein-70 gene for Cryptosporidium spp. The DNA amplicons were sequenced to determine genotypes and species. A mixed multivariable method was used to compare the presence of Giardia and Cryptosporidium in different stages of production. Both Giardia cysts and Cryptosporidium oocysts were present on all tested farms, with 50.8% of the samples positive for G. duodenalis and 44.3% positive for Cryptosporidium spp. by microscopy, and 66.4% and 55.7%, respectively, positive by polymerase chain reaction (PCR). No significant agreement was observed between microscopy and PCR method to detect Giardia and Cryptosporidium (p<0.05). The prevalence of Giardia in manure pits and finisher pigs did not differ (p>0.05), however, it was less frequent (odds ratio, OR=0.21 [0.07, 0.63]) among sows. Cryptosporidium was more likely (OR=3.6 [1.3, 9.9]) to be detected in manure pits and weaners (OR=3.3 [1.1, 10.0]) compared to finisher pigs, and it was less frequent (OR=0.06 [0.007, 0.55]) in sows than in finishers (p<0.05). DNA sequencing demonstrated that 92.1% of the Giardia isolates were Assemblage B and 7.9% were Assemblage E. The most prevalent Cryptosporidium were Cryptosporidium parvum (55.4%), and Cryptosporidium sp. pig genotype II (37.5%). These findings indicate that the occurrence of zoonotic isolates of G. duodenalis and Cryptosporidium is very high on swine farms in southern Ontario, and that there is a potential for transmission between swine and humans by means of cyst and oocyst contaminated water or foods.

Molecular Survey of Babesia microti in Wild Rodents in Central Croatia

Babesia divergens and B. divergens-like organisms are the main causative agents of human babesiosis in Europe. Recently, the first case of human infection with Babesia microti was confirmed in Germany, implicating the presence of zoonotic isolates. To estimate the presence of zoonotic B. microti in Croatia we analyzed 120 small wild mammals that serve as its reservoir by polymerase chain reaction. Yellow-necked mice (Apodemus flavicollis) and bank voles (Myodes glareolus) were both found to be infected with prevalence of 16.2%. Sequence analysis of the portion of 18S rDNA gene demonstrated that six polymerase chain reaction-positive samples, detected in both rodent species, were identical to that of the human Jena/Germany strain (EF413181). The other two isolates were identical to the nonzoonotic Munich strain (AB071177). The results of this study indicate the presence of zoonotic B. microti in A. flavicollis and M. glareolus in Croatia and a potential risk for human health.

Structural Basis for Potent Cross-Neutralizing Human Monoclonal Antibody Protection against Lethal Human and Zoonotic Severe Acute Respiratory Syndrome Coronavirus Challenge

Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002, and detailed phylogenetic and epidemiological analyses have suggested that it originated from animals. The spike (S) glycoprotein has been identified as a major component of protective immunity, and 23 different amino acid changes were noted during the expanding epidemic. Using a panel of SARS-CoV recombinants bearing the S glycoproteins from isolates representing the zoonotic and human early, middle, and late phases of the epidemic, we identified 23 monoclonal antibodies (MAbs) with neutralizing activity against one or multiple SARS-CoV spike variants and determined the presence of at least six distinct neutralizing profiles in the SARS-CoV S glycoprotein. Four of these MAbs showed cross-neutralizing activity against all human and zoonotic S variants in vitro, and at least three of these were mapped in distinct epitopes using escape mutants, structure analyses, and competition assays. These three MAbs (S109.8, S227.14, and S230.15) were tested for use in passive vaccination studies using lethal SARS-CoV challenge models for young and senescent mice with four different homologous and heterologous SARS-CoV S variants. Both S227.14 and S230.15 completely protected young and old mice from weight loss and virus replication in the lungs for all viruses tested, while S109.8 completely protected mice from weight loss and clinical signs in the presence of viral titers. We conclude that a single human MAb can confer broad protection against lethal challenge with multiple zoonotic and human SARS-CoV isolates, and we identify a robust cocktail formulation that targets distinct epitopes and minimizes the likely generation of escape mutants.

Predominance of subtype 3 among Blastocystis isolates from a major hospital in Singapore

Blastocystis is an enteric protozoan parasite commonly found in humans and animals. Phylogenetic and genotypic analyses have shown that Blastocystis exhibits extreme genetic diversity, and humans are host to a number of zoonotic isolates. In the present study, the prevalence of Blastocystis in 276 stool samples from a hospital in Singapore was examined, and for the first time, riboprinting using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to determine the genetic diversity of the Blastocystis isolated from the Singapore population. The prevalence rate was determined to be 3.3% (9/276), and Blastocystis displaying two main ribotypes were isolated. As a comparison, we performed PCR-RFLP using two different published methodologies, and both methods allowed the isolates to be divided into two distinct groups based on their riboprint patterns. According to a recently proposed classification scheme, 78% (7/9) of the isolates were of subtype 3, while 22% (2/9) were subtype 1. The predominance of subtype 3 in an urbanized city state such as Singapore is in agreement with the idea that subtype 3 is a genotype of human origin.