Population structure and virulence gene profiles of Streptococcus agalactiae collected from different hosts worldwide.

Streptococcus agalactiae is a leading cause of morbidity and mortality among neonates and causes severe infections in pregnant women and nonpregnant predisposed adults, in addition to various animal species worldwide. Still, information on the population structure of S. agalactiae and the geographical distribution of different clones is limited. Further data are urgently needed to identify particularly successful clones and obtain insights into possible routes of transmission within one host species and across species borders. We aimed to determine the population structure and virulence gene profiles of S. agalactiae strains from a diverse set of sources and geographical origins. To this end, 373 S. agalactiae isolates obtained from humans and animals from five different continents were typed by DNA microarray profiling. A total of 242 different S. agalactiae strains were identified and further analyzed. Particularly successful clonal lineages, hybridization patterns, and strains were identified that were spread across different continents and/or were present in more than one host species. In particular, several strains were detected in both humans and cattle, and several canine strains were also detected in samples from human, bovine, and porcine hosts. The findings of our study suggest that although S. agalactiae is well adapted to various hosts including humans, cattle, dogs, rodents, and fish, interspecies transmission is possible and occurs between humans and cows, dogs, and rabbits. The virulence and resistance gene profiles presented enable new insights into interspecies transmission and make a crucial contribution to the identification of suitable targets for therapeutic agents and vaccines.

Cytauxzoon europaeus infections in domestic cats in Switzerland and in European wildcats in France: a tale that started more than two decades ago.

BACKGROUND: Cytauxzoon spp. infection is believed to be a newly emerging tick-borne disease in felids in Europe, with three species of the haemoparasite having recently been differentiated in wild felids. In Switzerland, rare infections have been documented in domestic cats in the west and northwest of the country, the first of which was in 2014. The aims of the present study were: (i) to characterize a Cytauxzoon spp. hotspot in domestic cats in central Switzerland; (ii) to elucidate the geographic distribution of Cytauxzoon spp. in domestic cats in Switzerland; (iii) to assess suspected high-risk populations, such as stray and anaemic cats; and (iv) to investigate the newly emerging nature of the infection. Cytauxzoon spp. were further differentiated using mitochondrial gene sequencing. METHODS: The overall study included samples from 13 cats from two households in central Switzerland (study A), 881 cats from all regions of Switzerland (study B), 91 stray cats from a hotspot region in the northwest of Switzerland and 501 anaemic cats from across Switzerland (study C), and 65 Swiss domestic cats sampled in 2003 and 34 European wildcats from eastern France sampled in the period 1995-1996 (study D). The samples were analysed for Cytauxzoon spp. using real-time TaqMan quantitative PCR, and positive samples were subjected to 18S rRNA, cytochrome b (CytB) and cytochrome c oxidase subunit I (COI) gene sequencing. RESULTS: In study A, six of 13 cats from two neighbouring households in central Switzerland tested postive for Cytauxzoon spp.; two of the six infected cats died from bacterial infections. In studies B and C, only one of the 881 cats (0.1%; 95% confidence interval [CI]: 0-0.3%) in the countrywide survey and one of the 501 anaemic cats (0.2%; 95% CI: 0-0.6%) tested postive for Cytauxzoon spp. while eight of the 91 stray cats in the northwest of Switzerland tested positive (8.8%; 95% CI: 3.0-14.6%). In study D, Cytauxzoon spp. was detected in one of the 65 domestic cat samples from 2003 (1.5%; 95% CI: 0-4.5%) and in ten of the 34 European wildcat samples from 1995 to 1996 (29%; 95% CI: 14.2-44.7%). The isolates showed ≥ 98.6% sequence identities among the 18S rRNA, CytB and COI genes, respectively, and fell in the subclade Cytauxzoon europaeus based on CytB and COI gene phylogenetic analyses. CONCLUSIONS: The study challenges the newly emerging nature of Cytauxzoon spp. in central Europe and confirms that isolates from domestic cats in Switzerland and European wild felids belong to the same species.

Correction to: The absence of the drhm gene is not a marker for human-pathogenicity in European Anaplasma phagocytophilum strains.

An amendment to this paper has been published and can be accessed via the original article.

The absence of the drhm gene is not a marker for human-pathogenicity in European Anaplasma phagocytophilum strains.

BACKGROUND: Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophil granulocytes. It is transmitted by ticks of the Ixodes ricinus complex and causes febrile illness in humans and animals. The geographical distribution of A. phagocytophilum spans the Americas, Europe, Africa and Asia. However, human disease predominantly occurs in North America but is infrequently reported from Europe and Asia. In North American strains, the absence of the drhm gene has been proposed as marker for pathogenicity in humans whereas no information on the presence or absence of the drhm gene was available for A. phagocytophilum strains circulating in Europe. Therefore, we tested 511 European and 21 North American strains for the presence of drhm and compared the results to two other typing methods: multilocus sequence typing (MLST) and ankA-based typing. RESULTS: Altogether, 99% (478/484) of the analyzable European and 19% (4/21) of the North American samples from different hosts were drhm-positive. Regarding the strains from human granulocytic anaplasmosis cases, 100% (35/35) of European origin were drhm-positive and 100% (14/14) of North American origin were drhm-negative. Human strains from North America and Europe were both part of MLST cluster 1. North American strains from humans belonged to ankA gene clusters 11 and 12 whereas European strains from humans were found in ankA gene cluster 1. However, the North American ankA gene clusters 11 and 12 were highly identical at the nucleotide level to the European cluster 1 with 97.4% and 95.2% of identity, respectively. CONCLUSIONS: The absence of the drhm gene in A. phagocytophilum does not seem to be associated with pathogenicity for humans per se, because all 35 European strains of human origin were drhm-positive. The epidemiological differences between North America and Europe concerning the incidence of human A. phagocytophilum infection are not explained by strain divergence based on MLST and ankA gene-based typing.

Seroprevalence, biogeographic distribution and risk factors for Aelurostrongylus abstrusus infections in Swiss cats.

The metastrongyloid nematode Aelurostrongylus abstrusus is a worldwide occurring feline lungworm. The spectrum of clinical signs in infected cats ranges from mild (e.g. nasal discharge or cough) to severe respiratory distress. The aim of this seroepidemiological study was to define prevalence and risk factors for A. abstrusus infections in Swiss cats, to assess the biogeographic distribution and to investigate the influence of temperature and altitude on the occurrence of this parasite. Sera of 4067 domestic cats were collected from all over Switzerland, tested for the presence of antibodies against A. abstrusus by a novel ELISA and the results correlated with biogeographic aspects. A subsample of 1000 datasets was used for risk factor analyses. Overall, 10.7% (434/4067, 95% confidence intervals [CI]: 9.7-11.7%) of the cats were tested positive, with variations from 0.0% to 20.0% among ten different biogeographic regions. Differences were significant between the Western (13.9%, CI: 11.4-16.7%) and the Eastern (9.2%, CI: 8.0-10.5%) Swiss Plateau, possibly attributable to the suitability of the areas for intermediate hosts. In total 90.3% (392/434) of the seropositive cats originated from regions lower than 700 m above sea level. Correspondingly, 98.9% (429/434) of positive samples were obtained from regions with a mean temperature higher than -2 °C in January, suggesting altitude and temperature being limiting factors for A. abstrusus infections in Switzerland. Concerning individual risk factors, prevalence was higher in intact (15.5%, CI: 9.5-23.4%) than in neutered cats (5.8%, CI: 7.9-10.4%). Young adult cats (aged 11-22 months) were significantly more often seropositive (10/76, 13.2%, CI: 6.5-22.9%) than kittens aged 1-10 months (1/34, 2.9%, CI: 0.1-15.3%) or adult and senior cats > 22 months (58/889, 6.5%, CI: 5-8.4%). Outdoor cats and cats presenting respiratory signs tend to be more often positive than indoor cats (p = 0.077) and animals without respiratory signs (p = 0.086), respectively. We here confirm that the use of a serological test can contribute to improve the identification of infected animals, through evaluation of risk factors on a population level and for a better management on an individual level, overcoming the challenges represented by faecal examinations and the correlated underestimation of the occurrence of A. abstrusus in cats.

Genetic characterization of Anaplasma phagocytophilum strains from goats (Capra aegagrus hircus) and water buffalo (Bubalus bubalis) by 16S rRNA gene, ankA gene and multilocus sequence typing.

Anaplasma phagocytophilum is a Gram-negative obligate intracellular bacterium that replicates in neutrophil granulocytes. It is transmitted by ticks and causes tick-borne fever in domestic ruminants such as sheep, cattle and goats. However, in contrast to sheep and cattle little is known about the clinical course of infection in goats. We report here on three cases of symptomatic infection with A. phagocytophilum in two goats (Capra aegagrus hircus) and one water buffalo (Bubalus bubalis). The animals showed symptoms and laboratory findings similar to sheep and cattle. To our knowledge, this is the first report on the symptomatic infection of water buffalos with A. phagocytophilum. The infecting strains were genetically characterized by 16S rRNA gene, ankA gene and multilocus sequence typing (MLST). Four other strains from asymptomatically infected goats were also included. The ankA sequences from five goats were part of the formerly described ankA gene clusters I and IV that are known to contain A. phagocytophilum strains from sheep and cattle. However, the sequences from one goat and from the water buffalo belonged to ankA gene cluster II that was formerly described to be restricted to roe deer. A similar observation was made for MLST as three goats clustered with sequences from sheep and cattle, whereas three other goats and the water buffalo were found to be part of the roe deer cluster. However, since most of the strains from sheep and cattle were distinct from the roe deer strains, roe deer might not represent major reservoir hosts for tick-borne fever in domestic ruminants. When differing parts of the 16S rRNA gene were used for typing the results were conflicting. This shows that the use of a standardized typing method such as MLST is highly desirable to generate easily comparable results.

Human polymorphonuclear neutrophils are recruited by porcine chemokines acting on CXC chemokine receptor 2, and platelet-activating factor.

BACKGROUND: Pig-to-human xenotransplantation is hampered by strong humoral and cellular immune responses, including acute vascular rejection (AVR). Infiltration of vascular xenografts by recipient polymorphonuclear neutrophils (PMN) is an early feature of AVR. Since little is known about the initiation of PMN recruitment, the present study investigated whether activated porcine endothelial cells (EC) release factors that induce human PMN recruitment. METHODS: Primary and immortalized porcine aortic EC cultures were stimulated with phorbol-myristate acetate/ionomycin, lipopolysaccharide, tumor-necrosis factor-alpha, or interferon-gamma. The interleukin (IL)-8 concentration of porcine EC supernatants was tested by ELISA. Human and porcine PMN were isolated from peripheral blood by Ficoll sedimentation and centrifugation, characterized by morphology and flow cytometry, and analyzed for chemotaxis using Boyden chambers or Transwells. PMN chemokine receptor desensitization was determined by intracellular calcium-flux measurements. RESULTS: Porcine EC supernatants contained significant amounts of porcine IL-8 and triggered chemotaxis in both human and porcine PMN. Chemotaxis of porcine, but not human, PMN was inhibited by anti-porcine IL-8 antibodies and recombinant porcine IL-8 induced strong chemotaxis only in porcine PMN. Porcine EC supernatants desensitized human PMN CXC-chemokine receptor (CXCR) 2, but not CXCR1, a receptor for human IL-8. Human PMN chemotaxis induced by porcine EC supernatants was significantly inhibited by blocking CXCR2 and platelet-activating factor (PAF). CONCLUSIONS: Both chemokines acting via CXCR2 and PAF are released by porcine EC inducing efficient chemotaxis of human PMN. These mechanisms responsible for the recruitment of human PMN to porcine endothelium during cell-mediated rejection of xenografts represent potential targets for preventive strategies.

Rolling adhesion of human NK cells to porcine endothelial cells mainly relies on CD49d-CD106 interactions.

BACKGROUND: Acute vascular rejection in pig-to-primate xenotransplantation involves recognition and damage of porcine (po) endothelial cells (EC) by human (hu) leukocytes, probably including natural killer (NK) cells. To study such interactions we analyzed rolling and static adhesion of hu NK cells to po EC. METHODS: The effects of blocking hu and po adhesion molecules on the adhesion hu NK cells to po EC monolayers was analyzed under shear stress (10 min, 37 degrees C, 0.7 dynes/cm2) or under static conditions (10 min, 37 degrees C). All used cell populations were phenotypically characterized by flow cytometry. RESULTS: Blocking of CD106 on po EC or its ligand CD49d on hu NK cells decreased rolling adhesion of both fresh and activated hu NK cells by more than 75%. Masking of CD62L on fresh but not activated hu NK resulted in a 44% decrease in rolling adhesion, in line with the diminished cell surface expression of CD62L upon activation. Antibodies to CD31, CD54, CD62E, and CD62P on EC or CD11a, CD18, and CD162 on NK cells had only minor effects on rolling adhesion. The adhesion of the FcgammaRIII- hu NK cell line NK92 to po EC was inhibited by 95% after masking po CD106 whereas antibodies to po CD31, CD54, CD62E, or CD62P had no effect, thereby excluding effects of Fc-receptor-dependent binding of hu NK cells to po EC. Static adhesion of activated NK cells was reduced by approximately 60% by blocking either CD49d or CD106, by 47% by blocking CD11a, and by 82% upon simultaneous blocking of CD11a and CD49d. CONCLUSIONS: Interactions between hu CD49d and po CD106 are crucial for both rolling and firm adhesion of hu NK cells to po EC and thus represent attractive targets for specific therapeutic interventions to prevent NK cell-mediated responses against po xenografts.

Questing Dermacentor reticulatus harbouring Babesia canis DNA associated with outbreaks of canine babesiosis in the Swiss Midlands.

In 2011 and 2012, outbreaks of clinical canine babesiosis were observed in 2 areas of the Swiss Midlands that had no history of this disease so far. In one area, cases of canine babesiosis occurred over 2 consecutive tick seasons. The outbreaks involved 29 dogs, 4 of which died. All dogs were infected with large Babesia sp. as diagnosed in Giemsa-stained blood smears and/or PCR. These were identified as B. canis (formerly known as B. canis canis) by subsequent partial sequencing of the 18S rRNA gene of Babesia sp. Interestingly, the sequence indicated either a genotype with heterogeneity in the ssrRNA gene copies or double infection with different B. canis isolates. None of the dogs had a recent travel history, but one had frequently travelled to Hungary and had suffered twice from clinical babesiosis 18 and 24 months prior to the outbreak in autumn 2011. Retrospective sequencing of a stored blood DNA sample of this dog revealed B. canis, with an identical sequence to the Babesia involved in the outbreaks. For the first time in Switzerland, the partial 18S rRNA gene of B. canis could be amplified from DNA isolated from 19 out of 23 adult Dermacentor reticulatus ticks flagged in the same area. The sequence was identical to that found in the dogs. Furthermore, one affected dog carried a female D. reticulatus tick harbouring B. canis DNA. Our findings illustrate that, under favourable biogeographic and climatic conditions, the life-cycle of B. canis can relatively rapidly establish itself in previously non-endemic areas. Canine babesiosis should therefore always be a differential diagnosis when dogs with typical clinical signs are presented, regardless of known endemic areas.

Human Fas-ligand expression on porcine endothelial cells does not protect against xenogeneic natural killer cytotoxicity.

Several human leukocyte subsets including natural killer (NK) cells, cytotoxic T lymphocytes (CTL), and polymorphonuclear neutrophils (PMN) participate in cellular immune responses directed against vascularized pig-to-human xenografts. As these leukocytes express the death receptor Fas either constitutively (PMN) or upon activation (NK, CTL), we explored in vitro whether the transgenic expression of Fas ligand (FasL) on porcine endothelial cells (EC) is a valuable strategy to protect porcine xenografts. The porcine EC line 2A2 was stably transfected with human FasL (2A2-FasL) and interactions of 2A2-FasL with human leukocytes were analyzed using functional assays for apoptosis, cytotoxicity, chemotaxis, adhesion under shear stress, and transmigration. FasL expressed on porcine EC induced apoptosis in human NK and T cells, but did not protect porcine EC against killing mediated by human NK cells. 2A2-FasL released soluble FasL, which induced strong chemotaxis in human PMN. Adhesion under shear stress of PMN on 2A2-FasL cells was increased whereas transendothelial migration was decreased. In contrast, FasL had no effect on the adhesion of NK cells but increased their transmigration through porcine EC. Although FasL expression on porcine EC is able to induce apoptosis in human effector cells, it did not provide protection against xenogeneic cytotoxicity. The observed impact of FasL on adhesion and transendothelial migration provides evidence for novel biological functions of FasL.

The production of macrophage inflammatory protein-2 induced by soluble intercellular adhesion molecule-1 in mouse astrocytes is mediated by src tyrosine kinases and p42/44 mitogen-activated protein kinase.

Severe traumatic brain injury stimulates the release of soluble intercellular adhesion molecule-1 (sICAM-1) into CSF. Studies in cultured mouse astrocytes suggest that sICAM-1 induces the production of macrophage inflammatory protein-2 (MIP-2). In the present study, we investigated the underlying mechanisms for MIP-2 induction. sICAM-1 induced MIP-2 in astrocytes lacking membrane-bound ICAM-1, indicating that its action is due to heterophilic binding to an undescribed receptor rather than homophilic binding to surface ICAM-1. Signal transduction may be mediated by src tyrosine kinases, as the src tyrosine kinase inhibitors herbimycin A and PP2 abolished MIP-2 induction by sICAM-1. Phosphorylation of p42/44 mitogen-activated protein kinase (MAPK), but not of p38 MAPK, occurred further downstream, as evidenced by western blot analysis combined with the use of herbimycin A and specific MAPK inhibitors. By contrast, induction of MIP-2 by tumour necrosis factor-alpha (TNF-alpha) involved both p42/44 MAPK and p38 MAPK. Following stimulation with either sICAM-1 or TNF-alpha, astrocyte supernatants promoted chemotaxis of human neutrophils and incubation of these supernatants with anti-MIP-2 antibodies more efficiently suppressed the migration induced by sICAM-1 than by TNF-alpha. These results show that sICAM-1 induces the production of biologically active MIP-2 in astrocytes by heterophilic binding to an undefined receptor and activation of src tyrosine kinases and p42/44 MAPK.