Petra Griekspoor

Sampling for low-pathogenic avian influenza A virus in wild Mallard ducks: oropharyngeal versus cloacal swabbing.

Sampling for low-pathogenic avian influenza A virus in wild Mallard ducks : oropharyngeal versus cloacal swabbing

Marked host specificity and lack of phylogeographic population structure of Campylobacter jejuni in wild birds

Zoonotic pathogens often infect several animal species, and gene flow among populations infecting different host species may affect the biological traits of the pathogen including host specificity, transmissibility and virulence. The bacterium Campylobacter jejuni is a widespread zoonotic multihost pathogen, which frequently causes gastroenteritis in humans. Poultry products are important transmission vehicles to humans, but the bacterium is common in other domestic and wild animals, particularly birds, which are a potential infection source. Population genetic studies of C. jejuni have mainly investigated isolates from humans and domestic animals, so to assess C. jejuni population structure more broadly and investigate host adaptation, 928 wild bird isolates from Europe and Australia were genotyped by multilocus sequencing and compared to the genotypes recovered from 1366 domestic animal and human isolates. Campylobacter jejuni populations from different wild bird species were distinct from each other and from those from domestic animals and humans, and the host species of wild bird was the major determinant of C. jejuni genotype, while geographic origin was of little importance. By comparison, C. jejuni differentiation was restricted between more phylogenetically diverse farm animals, indicating that domesticated animals may represent a novel niche for C. jejuni and thereby driving the evolution of those bacteria as they exploit this niche. Human disease is dominated by isolates from this novel domesticated animal niche.

Prevalence of Campylobacter in wild birds of the mid-Atlantic region, USA.

We evaluated the occurrence of three Campylobacter species—C. jejuni, C. coli, and C. lari—from 333 wild bird fecal samples collected at Tri-State Bird Rescue and Research in Newark, Delaware, in 2008. Using multiplex polymerase chain reaction, we detected C. jejuni from six avian families with an overall prevalence rate of 7.2%. We did not detect any other Campylobacter species. Cam-pylobacter jejuni prevalence ranged widely between different avian families with crows (Corvidae) and gulls (Laridae) having the highest prevalence rates (23% and 25%, respectively).

Multilocus sequence typing of Campylobacter jejuni from broilers

Campylobacter jejuni isolates from a national Swedish Campylobacter monitoring in broilers were characterized by multilocus sequencing typing (MLST) in order to study the genetic diversity of this bacterial population. Isolates were initially characterized by pulsed-field gel electrophoresis (PFGE). One hundred were chosen for MLST genotyping. PFGE identified 69 distinct types compared to 44 different sequence types (STs) identified with MLST. Eighteen STs had not been described previously, while the remaining 26 STs were assigned to previously known clonal complexes. The majority of isolates were of genotypes noted in broilers and in humans in earlier studies. However, three clonal complexes, ST-206 complex, ST-677 complex and ST-1034 complex, previously associated with wild bird and environmental samples, were among the genotypes found. This study shows that most of the Swedish broiler isolates were of genotypes noted as common in broilers. However, it also highlights the potential influence of environmental sources on the broiler C. jejuni genotypes.

Campylobacter jejuni colonization in wild birds: Results from an infection experiment.

Campylobacter jejuni is a common cause of bacterial gastroenteritis in most parts of the world. The bacterium has a broad host range and has been isolated from many animals and environments. To investigate shedding patterns and putative effects on an avian host, we developed a colonization model in which a wild bird species, the European Robin Erithacus rubecula, was inoculated orally with C. jejuni from either a human patient or from another wild bird species, the Song Thrush Turdus philomelos. These two isolates were genetically distinct from each other and provoked very different host responses. The Song Thrush isolate colonized all challenged birds and colonization lasted 6.8 days on average. Birds infected with this isolate also showed a transient but significant decrease in body mass. The human isolate did not colonize the birds and could be detected only in the feces of the birds shortly after inoculation. European Robins infected with the wild bird isolate generated a specific antibody response to C. jejuni membrane proteins from the avian isolate, which also was cross-reactive to membrane proteins of the human isolate. In contrast, European Robins infected with the human isolate did not mount a significant response to bacterial membrane proteins from either of the two isolates. The difference in colonization ability could indicate host adaptations.

Campylobacter jejuni in penguins, Antarctica.

To the Editor: The wildlife of Antarctica is highly specialized. Although large animal species are limited primarily to penguins and seals, each species is often abundant. The high degree of isolation potentially protects Antarctic wildlife from diseases distributed in other areas of the world (1,2). Despite Antarctica’s isolation, however, human- or animal-related pathogens have been found there, or in the sub-Antarctic islands. For instance, serologic evidence of influenza virus A infections in penguins has been found (3), and both Salmonella spp. and Mycobacterium tuberculosis have been isolated from sub-Antarctic and Antarctic animals (4,5). Campylobacter jejuni is a leading cause of bacterial gastroenteritis in humans worldwide; it is usually found in the intestinal tract of various farm and wild animals, particularly birds (6,7).We previously reported finding 3 C. jejuni subsp. jejuni isolates in macaroni penguins (Eudyptes chrysolophus; Figure) from Bird Island (54°00′S, 38°02′W), South Georgia (1). Phenotypic tests and 16S rRNA gene sequencing showed that the penguin isolates were identical to each other, and macrorestriction profiling of pulsed-field gel electrophoresis fragments showed that they were very similar to fragments isolated from poultry in Washington in 1984 (1). Because the isolates were retrieved from macaroni penguin chicks, we concluded that the animals had acquired the infection locally and that this was likely an instance of introduction of a pathogen to the Antarctic region. Figure Macaroni penguins (Eudyptes chrysolophus). Photo by Jonas Bonnedahl. However, restriction fragment pattern resemblance is not identical to genetic relatedness and, given the relevance of the question of origin, this resemblance led us to use a new method for genetic characterization. We reanalyzed the macaroni penguin isolates with multilocus sequence typing (MLST), a method that uses sequence data from 7 unlinked loci for genetic identification (8), complemented with flaA gene sequencing. A benefit of this method is the increasing availability of epidemiologic databases in which isolates can be compared (e.g., http://pubmlst.org/cam,pylobacter). The isolates were thawed and cultured on conventional blood agar (Columbia agar II containing 8% [vol/vol] whole horse blood) at 42°C in a microaerobic gas environment, with the CampyGen gas-generating system (CN0025A; Oxoid Ltd, Basingstoke, UK) and the BBL GasPak system (BD, Franklin Lakes, NJ, USA). Bacterial DNA was prepared by making a suspension of freshly grown bacterial cells in 200 µL of phosphate-buffered saline (Sigma, St. Louis, MO, USA). Genomic DNA was extracted by use of a Bio Robot M48 (QIAGEN, Hilden, Germany) with a MagAttract DNA mini M48 kit, according to the instructions of the manufacturer. The PCR amplification and nucleotide sequencing followed the original protocol in principle (8). The amplification products were purified and sequenced by using internal separated nested primer pairs. The 3 isolates from macaroni penguins were all of the same genotype (sequence type [ST]–45) and thus have a common origin. The ST-45 sequence type is the central genotype of the ST-45 clonal complex, a complex often associated with human disease and asymptomatic infection in poultry (9,10). Indeed, nearly 42% of the ST-45 samples available in the MLST database have been isolated from humans (31% from poultry), and similar percentages have been observed for the ST-45 clonal complex as a whole. The ST-45 clonal complex is large (composed of 195 individual STs) and has been isolated to date from a variety of environmental sources and different geographic regions, with the exception of the Arctic. The isolates were identical at the flaA locus, all having the allele 21/peptide 2 designation (http://pubmlst.org/campylobacter/flaA). This particular peptide is found in 31 records in the database and is thus not unique to the penguin isolates. Our MLST analysis confirms that the C. jejuni isolates from the penguins were of a genotype common among humans with disease and among our food animals. C. jejuni is not normally distributed among Antarctic animals (1,2), which indicates that this strain may have been imported through human activities. On Bird Island, such activities were carried out by scientists at the British Antarctic Survey base. At the time of the study, toilet wastes from the station were emptied into the surrounding waters, providing a possible transmission route for human-associated C. jejuni to reach wildlife, including penguins. Other possible sources of the C. jejuni infections include wastes from passing ships or seabirds that pick up the bacteria during offshore feeding excursions (for albatrosses, these can be ≈1,000 km). Once established in a penguin colony, a gastrointestinal pathogen may be transmitted rapidly among individual birds as they are breeding densely and producing a large amount of feces (guano) in the colony. C. jejuni infection in birds is normally not associated with overt disease, but other and possibly more devastating pathogens introduced to Antarctic animals could potentially cause outbreaks.

Amoebae and algae can prolong the survival of Campylobacter species in co-culture

Several species of free-living amoebae can cause disease in humans. However, in addition to the direct pathogenicity of e.g. Acanthamoebae and Naegleria species, they are recognized as environmental hosts, indirectly involved in the epidemiology of many pathogenic bacteria. Although several studies have demonstrated intracellular survival of many different bacteria in these species, the extent of such interactions as well as the implications for the epidemiology of the bacterial species involved, are largely unknown and probably underestimated. In this study, we evaluated eight different unicellular eukaryotic organisms, for their potential to serve as environmental hosts for Campylobacter species. These organisms include four amoebozoas (Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba rhysodes and Hartmanella vermiformis), one alveolate (Tetrahymena pyriformis), one stramenopile (Dinobryon sertularia), one eugoenozoa (Euglena gracilis) and one heterolobosea (Naegleria americana). Campylobacter spp. including Campylobacter jejuni, Campylobacter coli and Campylobacter lari are the most common cause of gastroenteritis in the western world. Survival and replication of these three species as well as Campylobacter hyointestinalis were assessed in co-cultures with the eukaryotic organisms. Campylobacter spp. generally survived longer in co-cultures, compared to when incubated in the corresponding growth media. The eukaryotic species that best promoted bacterial survival was the golden algae D. sertularia. Three species of amoebozoas, of the genus Acanthamoeba promoted both prolonged survival and replication of Campylobacter spp. The high abundance in lakes, ponds and water distribution networks of these organisms indicate that they might have a role in the epidemiology of campylobacteriosis, possibly contributing to survival and dissemination of these intestinal pathogens to humans and other animals. The results suggest that not only C. jejuni, but a variety of Campylobacter spp. can interact with different eukaryotic unicellular organisms.

Genetic diversity and host associations in Campylobacter jejuni from human cases and broilers in 2000 and 2008

Campylobacter jejuni is an important food-borne pathogen, with a global distribution. It can colonize numerous host species, including both domestic and wild animals, but is particularly associated with birds (poultry and wild birds). For human campylobacteriosis, poultry products are deemed the most significant risk factor for acquiring infection. We conducted a genotyping and host attribution study of a large representative collection of C. jejuni isolated from humans and broilers in Sweden in the years 2000 and 2008. In total 673 broiler and human isolates from 10 different abattoirs and 6 different hospitals were genotyped with multilocus sequence typing. Source attribution analyses confirmed the strong linkage between broiler C. jejuni and domestic human cases, but also indicated a significant association to genotypes more commonly found in wild birds. Genotype distributions did not change dramatically between the two study years, suggesting a stable population of infecting bacteria.

Campylobacter jejuni sequence types show remarkable spatial and temporal stability in Blackbirds

Background The zoonotic bacterium Campylobacter jejuni has a broad host range but is especially associated with birds, both domestic and wild. Earlier studies have indicated thrushes of the genus Turdus in Europe to be frequently colonized with C. jejuni, and predominately with host-associated specific genotypes. The European Blackbird Turdus merula has a large distribution in Europe, including some oceanic islands, and was also introduced to Australia by European immigrants in the 1850s. Methods The host specificity and temporal stability of European Blackbird C. jejuni was investigated with multilocus sequence typing in a set of isolates collected from Sweden, Australia, and The Azores. Results Remarkably, we found that the Swedish, Australian, and Azorean isolates were genetically highly similar, despite extensive spatial and temporal isolation. This indicates adaptation, exquisite specificity, and stability in time for European Blackbirds, which is in sharp contrast with the high levels of recombination and mutation found in poultry-related C. jejuni genotypes. Conclusion The maintenance of host-specific signals in spatially and temporally separated C. jejuni populations suggests the existence of strong purifying selection for this bacterium in European Blackbirds.