First Report of Zoonotic Genotype of Giardia duodenalis in Mussels (Mytilus edulis) from Patagonia Argentina.

Blue mussels (Mytilus edulis) are among the most consumed fishery products globally. Foodborne outbreaks of mussel-associated infections by viral, bacterial, and parasitic pathogens have been reported in the last years. In this study, we investigated the occurrence, genetic diversity, and zoonotic potential of the diarrhea-causing enteric protozoan Giardia duodenalis in blue mussels from Caleta Córdova in Chubut Province, southeast Patagonia, Argentina. A total of 344 free-living blue mussels were collected and distributed in 53 aliquots of pooled mussel tissue (each containing 5‒7 specimens) during the period 2015‒2018. Conventional optical microscopy was used as screening method for the detection of G. duodenalis cysts in pooled, homogenized tissues. Samples with a positive result were assessed by a multilocus sequence genotyping scheme based on the amplification of partial fragments of the glutamate dehydrogenase and ß-giardin genes of the parasite. G. duodenalis cysts were found in 30.2% (16/53) of the aliquots of pooled mussel tissue tested. PCR and sequencing analyses revealed the presence of G. duodenalis subassemblage BIV in selected aliquots. To the best of our knowledge, this is the first description of zoonotic subassemblage BIV from blue mussels in Argentina.

Multilocus phylogeny and cryptic diversity of white-toothed shrews (Mammalia, Eulipotyphla, Crocidura) in China.

BACKGROUND: Crocidura, the most speciose mammalian genus, occurs across much of Asia, Europe and Africa. The taxonomy of Chinese representatives has been studied primarily based on cursory morphological comparisons and their molecular phylogenetic analyses remain unexplored. In order to understand the phylogeny of this group in China, we estimated the first multilocus phylogeny and conducted species delimitation, including taxon sampling throughout their distribution range. RESULTS: We obtained one mitochondrial gene (cytb) (~ 1, 134 bp) and three nuclear genes (ApoB, BRCA1, RAG1) (~ 2, 170 bp) for 132 samples from 57 localities. Molecular analyses identified at least 14 putative species that occur within two major well-supported groups in China. Polyphyletic C. wuchihensis appears to be composed of two putative species. Two subspecies, C. rapax rapax and C. rapax kurodai should be elevated to full species status. A phylogenetic tree based on mitochondrial gene from Asian Crocidura species showed that the C. rapax rapax is embedded within C. attenuata, making the latter a paraphyletic group. Three strongly supported undescribed species (C. sp.1, C. sp.2 and C. sp.3) are revealed from Zada County of Tibet (Western China), Hongjiang County of Hunan Province (Central China) and Dongyang County of Zhejiang Province (Eastern China), Motuo County of Tibet, respectively. The divergence time estimation suggested that China's Crocidura species began to diversify during the late Pliocene (3.66 Ma) and the Early Pleistocene (2.29 Ma), followed by a series of diversifications through the Pleistocene. CONCLUSIONS: The cryptic diversity found in this study indicated that the number of species is strongly underestimated under the current taxonomy. We propose that the three undescribed species should be evaluated using extensive taxon sampling and comprehensive morphological and morphometric approaches. Climate change since the late Pliocene and the uplift of the Qinghai-Tibet Plateau may result in the diversification and speciation of China's Crocidura species. In short, the underestimated diversity underlines the need for a taxonomic revision of Chinese Crocidura species.

First case report of fatal Nocardia nova infection in yellow-bibbed lory (Lorius chlorocercus) identified by multilocus sequencing.

BACKGROUND: Nocardiosis is often a multi-systemic disease in humans and other mammals. Nocardiosis in birds is uncommon. Laboratory identification of Nocardia to the species level is difficult by traditional phenotypic methods based on biochemical reactions and hydrolysis tests, and is most accurately performed by sequencing multiple gene targets. CASE PRESENTATION: We report the first case of fatal Nocardia nova infection in a yellow-bibbed lory nestling in an oceanarium diagnosed by multilocus sequencing. Necropsy examination showed effacement of normal sternal musculature with yellowish, firm aberrant material, and diffuse infiltration of the lungs with nodular, tan to yellow foci. Histologically, severe granulomatous inflammation with marked necrosis was observed in the lung, spleen and sternal musculature. Fine, sometimes Gram-positive, 0.5-1 µm wide, branching and beaded filamentous organisms were visible within the lesions. They were acid-fast on Fite-Faraco stain. Tissue samples obtained from the sternum, liver, right lung and right kidney recovered Nocardia species. Sequencing of four gene loci and phylogenetic analysis of concatenated (gyrB-16S-secA1-hsp65) sequences revealed that the isolate was N. nova. CONCLUSIONS: We report the first case of N. nova infection in yellow-bibbed lorry (Lorius chlorocercus). The present case is the first one of which the species identity of the isolate was determined by multilocus sequencing. Molecular diagnosis is important for identifying the Nocardia to species level and understanding the epidemiology of nocardiosis in birds.

Pathogenic variability among Pasteurella multocida type A isolates from Brazilian pig farms.

BACKGROUND: Pasteurella multocida type A (PmA) is considered a secondary agent of pneumonia in pigs. The role of PmA as a primary pathogen was investigated by challenging pigs with eight field strains isolated from pneumonia and serositis in six Brazilian states. Eight groups of eight pigs each were intranasally inoculated with different strains of PmA (1.5 mL/nostril of 10e7 CFU/mL). The control group (n = 12) received sterile PBS. The pigs were euthanized by electrocution and necropsied by 5 dpi. Macroscopic lesions were recorded, and swabs and fragments of thoracic and abdominal organs were analyzed by bacteriological and pathological assays. The PmA strains were analyzed for four virulence genes (toxA: toxin; pfhA: adhesion; tbpA and hgbB: iron acquisition) by PCR and sequencing and submitted to multilocus sequence typing (MLST). RESULTS: The eight PmA strains were classified as follows: five as highly pathogenic (HP) for causing necrotic bronchopneumonia and diffuse fibrinous pleuritis and pericarditis; one as low pathogenic for causing only focal bronchopneumonia; and two as nonpathogenic because they did not cause injury to any pig. PCR for the gene pfhA was positive for all five HP isolates. Sequencing demonstrated that the pfhA region of the HP strains comprised four genes: tpsB1, pfhA1, tpsB2 and pfhA2. The low and nonpathogenic strains did not contain the genes tpsB2 and pfhA2. A deletion of four bases was observed in the pfhA gene in the low pathogenic strain, and an insertion of 37 kb of phage DNA was observed in the nonpathogenic strains. MLST clustered the HP isolates in one group and the low and nonpathogenic isolates in another. Only the nonpathogenic isolates matched sequence type 10; the other isolates did not match any type available in the MLST database. CONCLUSIONS: The hypothesis that some PmA strains are primary pathogens and cause disease in pigs without any co-factor was confirmed. The pfhA region, comprising the genes tpsB1, tpsB2, pfhA1 and pfhA2, is related to the pathogenicity of PmA. The HP strains can cause necrotic bronchopneumonia, fibrinous pleuritis and pericarditis in pigs and can be identified by PCR amplification of the gene pfhA2.

Molecular phylogeny and biogeographic history of the Neotropical tribe Glandulocaudini (Characiformes: Characidae: Stevardiinae)

Although former studies on systematics and biogeography represent a progress on the knowledge of the tribe Glandulocaudini, none was grounded on molecular evidence. Thus, the first hypothesis of relationships for the tribe based on a multilocus analysis is presented, including all genera and most of the valid species. DNA sequences of Glandulocauda caerulea and Mimagoniates sylvicola were analyzed for the first time. A molecular clock analysis was used to estimate the origin of the Glandulocaudini and the approximate timing of cladogenetic events within the group. Glandulocaudini was recovered as monophyletic. No hypothesis recovered Glandulocauda as monophyletic, since G. melanopleura is sister to Lophiobrycon weitzmani while G. caerulea is closely related to Mimagoniates. The relationships within the latter genus were resolved. The molecular clock results indicate the origin of the Glandulocaudini during the Miocene with diversification in the group occurring from Neogene to Pleistocene. These results corroborated the hypothesis that its origin took place on the Brazilian crystalline shield with the subsequent occupation of the Atlantic Coastal drainages. Apparently, Pleistocene sea-level fluctuations might have shaped the distribution pattern of some species in Glandulocaudini.(AU)

Embora estudos prévios sobre sistemática e biogeografia representam um avanço no conhecimento da tribo Glandulocaudini, nenhum foi baseado em evidência molecular. Assim, a primeira hipótese de relações para a tribo com base em uma análise multilocus é apresentada, incluindo todos os gêneros e a maioria das espécies válidas. Sequências de DNA de Glandulocauda caerulea e Mimagoniates sylvicola foram analisadas pela primeira vez. Uma análise de relógio molecular foi utilizada para estimar a origem de Glandulocaudini e datas aproximadas de eventos cladogenéticos dentro do grupo. Glandulocaudini foi recuperada como monofilética. Nenhuma hipótese recuperou Glandulocauda como monofilético, uma vez que G. melanopleura é irmã de Lophiobrycon weitzmani e G. caerulea está proximamente relacionada a Mimagoniates. As relações dentro deste último gênero foram resolvidas. Os resultados do relógio molecular indicam que Glandulocaudini originou-se durante o Mioceno, com diversificação dentro do grupo ocorrendo desde o Neogeno até o Pleistoceno. Estes resultados corroboram a hipótese da sua origem no escudo cristalino brasileiro, com a subsequente ocupação das drenagens costeiras atlânticas. Aparentemente, as flutuações pleistocênicas do nível do mar podem ter moldado o padrão de distribuição de algumas espécies em Glandulocaudini.(AU)

First case report of non-human primates (Alouatta clamitans) with the hypervirulent Klebsiella pneumoniae serotype K1 strain ST 23: A possible emerging wildlife pathogen.

BACKGROUND: Hypervirulent strain of Klebsiella pneumoniae genotype K1 isolates have recently emerged, causing severe pyogenic liver abscess complicated by devastating metastatic infections in humans. METHODS: We describe a short outbreak of the non-human primate (NHP) research center, associated with a hypervirulent K. pneumoniae. The genetic similarity of the strains was evaluated by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) techniques, and virulence encoding genes were detected by polymerase chain reaction (PCR). RESULTS: The isolates were phenotypically like strains causing community-acquired invasive liver abscess syndrome in humans. All strains exhibited identical PFGE patterns and were found to belong to ST23 and presented a hypermucovisity phenotype and possessed magA and rmpA gene. CONCLUSION: This is the first case report of NHPs caused by K. pneumoniae displaying a hypermucoviscosity phenotype and belonging to capsular serotypes K1 and ST23.

Establishment of Canine-Derived Giardia duodenalis Isolates in Culture.

Researchers continue to rely on axenic cultivation of Giardia duodenalis trophozoites in vitro to study the life cycle and host-parasite interactions of G. duodenalis and to develop vaccines and drugs to prevent and treat giardiasis. The majority of in vitro studies of G. duodenalis have used a small subset of isolates, mostly of assemblage A, and these isolates are usually originally isolated from humans. The most commonly used isolate for lab studies is known as WB. Canine giardiasis is a disease of veterinary importance, but it may also be of relevance in zoonotic transmission. Few G. duodenalis isolates from dogs have been adapted to in vitro culture, probably because the methods used are not suitable for the canine-specific genotypes that tend to dominate in most dog populations. In the current study, an experimental approach to cultivating canine-derived isolates of G. duodenalis was attempted by modification of the standard protocol based on physiological differences between the human and canine digestive system. An adapted method is described for improving the rate of in vitro excystation of cysts isolated from dogs by chemically weakening the cyst wall. A new canine-derived assemblage A G. duodenalis isolate was successfully adapted to axenic culture by using this method; the dog apparently had a mixed infection of assemblages A and D, but the assemblage A successfully outcompeted the assemblage D under conditions of in vitro culture. Based on the results, reasons regarding why humans do not seem to be suitable hosts for G. duodenalis in assemblages C and D are discussed.

The utility of diversity profiling using Illumina 18S rRNA gene amplicon deep sequencing to detect and discriminate Toxoplasma gondii among the cyst-forming coccidia.

Next-generation sequencing (NGS) has the capacity to screen a single DNA sample and detect pathogen DNA from thousands of host DNA sequence reads, making it a versatile and informative tool for investigation of pathogens in diseased animals. The technique is effective and labor saving in the initial identification of pathogens, and will complement conventional diagnostic tests to associate the candidate pathogen with a disease process. In this report, we investigated the utility of the diversity profiling NGS approach using Illumina small subunit ribosomal RNA (18S rRNA) gene amplicon deep sequencing to detect Toxoplasma gondii in previously confirmed cases of toxoplasmosis. We then tested the diagnostic approach with species-specific PCR genotyping, histopathology and immunohistochemistry of toxoplasmosis in a Risso's dolphin (Grampus griseus) to systematically characterise the disease and associate causality. We show that the Euk7A/Euk570R primer set targeting the V1-V3 hypervariable region of the 18S rRNA gene can be used as a species-specific assay for cyst-forming coccidia and discriminate T. gondii. Overall, the approach is cost-effective and improves diagnostic decision support by narrowing the differential diagnosis list with more certainty than was previously possible. Furthermore, it supplements the limitations of cryptic protozoan morphology and surpasses the need for species-specific PCR primer combinations.

New insights on the genetic diversity of the honeybee parasite Nosema ceranae based on multilocus sequence analysis.

The microsporidian parasite Nosema ceranae is a common pathogen of the Western honeybee (Apis mellifera) whose variable virulence could be related to its genetic polymorphism and/or its polyphenism responding to environmental cues. Since the genotyping of N. ceranae based on unique marker sequences had been unsuccessful, we tested whether a multilocus approach, assessing the diversity of ten genetic markers ­ encoding nine proteins and the small ribosomal RNA subunit ­ allowed the discrimination between N. ceranae variants isolated from single A. mellifera individuals in four distant locations. High nucleotide diversity and allele content were observed for all genes. Most importantly, the diversity was mainly present within parasite populations isolated from single honeybee individuals. In contrast the absence of isolate differentiation precluded any taxa discrimination, even through a multilocus approach, but suggested that similar populations of parasites seem to infect honeybees in distant locations. As statistical evolutionary analyses showed that the allele frequency is under selective pressure, we discuss the origin and consequences of N. ceranae heterozygosity in a single host and lack of population divergence in the context of the parasite natural and evolutionary history.

Molecular typing of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) isolated from animals and retail meat in North Dakota, United States.

The objective of this study was to determine the prevalence and molecular typing of methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) in food-producing animals and retail meat in Fargo, North Dakota. A two-step enrichment followed by culture methods were used to isolate S. aureus from 167 nasal swabs from animals, 145 samples of retail raw meat, and 46 samples of deli meat. Positive isolates were subjected to multiplex polymerase chain reaction in order to identify the genes 16S rRNA, mecA, and Panton-Valentine Leukocidin. Pulsed-field gel electrophoresis and multilocus sequence typing were used for molecular typing of S. aureus strains. Antimicrobial susceptibility testing was carried out using the broth microdilution method. The overall prevalence of S. aureus was 37.2% (n=133), with 34.7% (n=58) of the animals positive for the organism, and the highest prevalence observed in pigs (50.0%) and sheep (40.6%) (p<0.05); 47.6% (n=69) of raw meat samples were positive, with the highest prevalence in chicken (67.6%) and pork (49.3%) (p<0.05); and 13.0% (n=6) of deli meat was positive. Five pork samples (7.0%) were positive for MRSA, of which three were ST398 and two were ST5. All exhibited penicillin resistance and four were multidrug resistant (MDR). The Panton-Valentine Leukocidin gene was not detected in any sample by multiplex polymerase chain reaction. The most common clones in sheep were ST398 and ST133, in pigs and pork both ST398 and ST9, and in chicken ST5. Most susceptible S. aureus strains were ST5 isolated from chicken. The MDR isolates were found in pigs, pork, and sheep. The presence of MRSA, MDR, and the subtype ST398 in the meat production chain and the genetic similarity between strains of porcine origin (meat and animals) suggest the possible contamination of meat during slaughtering and its potential transmission to humans.

Streptococcus equi subsp. zooepidemicus isolates from equine infectious endometritis belong to a distinct genetic group.

Streptococcus equi subsp. zooepidemicus is the pathogen most commonly isolated from the uterus of mares. S. zooepidemicus is an opportunistic pathogen and part of the resident flora in the caudal reproductive tract. The aim of this study was to investigate whether a genotypically distinct subpopulation of S. zooepidemicus is associated with endometritis in the mare, by genotyping and comparing uterine S. zooepidemicus strains with isolates from the vagina and clitoral fossa. Mares with (n=18) or without (n=11) clinical symptoms of endometritis were included. Uterine samples were obtained using a guarded endometrial biopsy punch, whereas a swab was used to recover samples from the cranial vagina and the clitoral fossa. If S. zooepidemicus was present, up to three colonies were selected from each anatomical location (max. 9 isolates per mare). Bacterial isolates were characterized by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). S. zooepidemicus was isolated from the endometrium of 12 mares. A total of 88 isolates were analyzed by PFGE: 31 from the endometrium, 26 from the cranial vagina and 31 isolates from the clitoral fossa. For MLST 21 isolates were chosen. Results demonstrated a higher genetic similarity of the isolates obtained from infectious endometritis compared to isolates obtained from the caudal reproductive tract. In conclusion, we demonstrate for the first time that a genetically distinct group of S. zooepidemicus is associated with infectious endometritis in the mare.

Detection and characterization of Histoplasma capsulatum in a German badger (Meles meles) by ITS sequencing and multilocus sequencing analysis.

A wild badger (Meles meles) with a severe nodular dermatitis was presented for post mortem examination. Numerous cutaneous granulomas with superficial ulceration were present especially on head, dorsum, and forearms were found at necropsy. Histopathological examination of the skin revealed a severe granulomatous dermatitis with abundant intralesional round to spherical yeast-like cells, 2-5 µm in diameter, altogether consistent with the clinical appearance of histoplasmosis farciminosi. The structures stained positively with Grocott's methenamine silver and Periodic acid-Schiff stains, but attempts to isolate the etiologic agent at 25 and 37°C failed. DNA was directly extracted from tissue samples and the ribosomal genes ITS1-5.8S-ITS2 were partially sequenced. This revealed 99% identity to sequences from Ajellomyces capsulatus, the teleomorph of Histoplasma capsulatum, which was derived from a human case in Japan, as well as from horses from Egypt and Poland. Phylogenetic multi-locus sequence analysis demonstrated that the fungus in our case belonged to the Eurasian clade which contains members of former varieties H. capsulatum var. capsulatum, H. capsulatum var. farciminosum. This is the first study of molecular and phylogenetic aspects of H. capsulatum, as well as evidence for histoplasmosis farciminosi in a badger, further illuminating the role of this rare pathogen in Central Europe.

Screening for methicillin-resistant staphylococci in dogs admitted to a veterinary teaching hospital.

This study investigated the nasal carriage of methicillin-resistant staphylococci (MRS) in dogs (n=177) prior to medical examination or surgery in a veterinary teaching hospital. Nasal swab samples were collected after induction of anaesthesia and incubated overnight in salt enriched trypticase-soy broth. Cultures were analysed on two different agar media containing cefoxitin. Suspected MRS isolates were genotypically identified and characterised by antimicrobial susceptibility testing and staphylococcal cassette chromosome mec (SCCmec)-typing. Methicillin-resistant Staphylococcus aureus (MRSA) isolates were additionally characterised by spa-typing and multilocus sequence typing. The presence of Panton-Valentine leukocidin (pvl) genes was determined by PCR. MRS carriage was compared between animals with or without an infectious process. Two MRSA were isolated, both belonging to typical Belgian human hospital clones and lacking pvl. Additionally a methicillin-resistant Staphylococcus haemolyticus carrying a type V SCCmec was detected. No relationship was observed between MRS carriage and presence of infections. The results suggest that MRS are present in dogs originating from the community, albeit at a low prevalence. This could pose risks for cross contamination of dogs and their owners.