Estella Prukner-Radovčić

Chlamydial infections in feral pigeons in Europe: Review of data and focus on public health implications.

Feral pigeons (Columba livia domestica), which thrive in most European towns and cities, are commonly infected with the zoonotic bacterium Chlamydophila psittaci, the agent of psittacosis (also known as ornithosis) in humans. A number of surveys carried out over the last thirty years across Europe have detected high seropositivity values and high percentages of infection in feral pigeon populations. Overall, when considering data from 11 European countries, seropositivity values to C. psittaci in the sampled populations ranged from 19.4% to 95.6%. In most surveys, the complement fixation test was used, and antibodies were detected in 19.4-66.3% of the samples, with a median of 46.1%. Indirect immunofluorescence and ELISA tests were employed less frequently, but led to the detection of higher percentages of seropositivity (23.7-67.7% and 35.9-95.6%, respectively). Attempts to grow C. psittaci in cell culture or embryonated chicken eggs were successful in 2-42.3% and 0-57.1% of samples, respectively, antigen detection methods were positive in 2.3-40% of samples, while conventional PCR and real-time PCR using different genomic targets detected the organism in 3.4-50% of samples. Twenty-five C. psittaci isolates from pigeons were typed as ompA genotype B (n=14), E (n=10) and E/B (n=1). The huge increase of feral pigeon populations in Europe is a major cause of concern for the detrimental effect of pigeon droppings on environmental hygiene, in addition to the extensive damage due to the fouling of buildings and monuments. The most important pathogenic organism transmissible from feral pigeons to humans is C. psittaci, with 101 cases of disease reported in the literature. Exposure to C. psittaci-contaminated dust, direct contact with pigeons through handling and, to a lesser extent, through pigeon feeding have been identified as hazardous exposures in more than half of the human cases, while loose or transient contacts with feral pigeons have been mentioned in about 40% of the cases. Education initiatives as to the communication of a health risk resulting from contact with pigeons and pigeon excreta should primarily be targeted at individuals who may be exposed to C. psittaci-contaminated dust, such as demolition/construction workers. Recommendations to this category of workers include wearing protective clothes with hoods, boots, gloves and air filter face masks when removing pigeon faeces from roofs, garrets and buildings, especially if working indoors. Monitoring for C. psittaci infections in these workers over time should also be considered. Children should be warned not to handle sick or dead pigeons, and immunocompromised individuals should be advised to carefully limit their contact to feral pigeons. Culling of pigeons by shooting or poisoning is both unethical and ineffective as the place of the killed birds in the population is quickly filled by new juveniles or immigrating birds from neighbouring areas. Pigeon-deterring systems, such as nets and plastic or metal spikes applied to buildings and monuments will prevent their fouling, and the administration of contraceptive drugs may allow size regulation of the pigeon populations. Nevertheless, the measure that will ultimately lead to permanent reduction and will establish healthy sustainable populations is the restriction of indiscriminate feeding by pigeon lovers. The erection of dovecotes and artificial breeding facilities should be considered for providing shelter and a balanced diet to the birds, as well as a chance of interaction for pigeon lovers in a hygienically controlled environment.

Molecular characterization of atypical Chlamydia and evidence of their dissemination in different European and Asian chicken flocks by specific real-time PCR.

Chlamydia psittaci is a zoonotic pathogen associated primarily with avian chlamydiosis. New chlamydial agents with suspected zoonotic potential were recently detected from domestic poultry in Germany and France indicating that the spectrum of Chlamydiaceae encountered in birds is not confined to a single chlamydial species. For further characterization, a specific real-time PCR targeting the conserved 16S rRNA gene was developed and validated for a specific detection of these atypical Chlamydiaceae. In order to address the epidemiological importance of the new chlamydial agents and their distribution, Chlamydiaceae-positive chicken samples collected from flocks from five different countries were examined. The results confirmed that C.psittaci is not the predominant chlamydial species among chickens examined and suggested that the new chlamydial agents could putatively be widespread in poultry flocks (France, Greece, Croatia, Slovenia and China at least) justifying their systematic investigation when poultry samples are submitted to laboratories for avian chlamydiosis diagnosis. Besides, 16S rRNA-based dendrogram, including sequences from both isolates of the new chlamydial agents or positive samples as well as representative sequences from species belonging to the order Chlamydiales, showed the new chlamydial agents to form a distinct line of descent separated from those of other chlamydial species, but clearly grouped within the family Chlamydiaceae. Finally, the phylogenetic tree inferred from the multi-locus sequence typing based on four housekeeping fragments (gatA, gidA, enoA and hflX) and the ompA-based dendrogram showed an almost identical topology of the new chlamydial agents with that recovered by 16S rRNA-based dendrogram. Interestingly, partial ompA gene sequences displayed considerable diversity among isolates.

Characterization of Egg Laying Hen and Broiler Fecal Microbiota in Poultry Farms in Croatia, Czech Republic, Hungary and Slovenia

Poultry meat is the most common protein source of animal origin for humans. However, intensive breeding of animals in confined spaces has led to poultry colonisation by microbiota with a zoonotic potential or encoding antibiotic resistances. In this study we were therefore interested in the prevalence of selected antibiotic resistance genes and microbiota composition in feces of egg laying hens and broilers originating from 4 different Central European countries determined by real-time PCR and 16S rRNA gene pyrosequencing, respectively. strA gene was present in 1 out of 10,000 bacteria. The prevalence of sul1, sul2 and tet(B) in poultry microbiota was approx. 6 times lower than that of the strA gene. tet(A) and cat were the least prevalent being present in around 3 out of 10,000,000 bacteria forming fecal microbiome. The core chicken fecal microbiota was formed by 26 different families. Rather unexpectedly, representatives of Desulfovibrionaceae and Campylobacteraceae, both capable of hydrogen utilisation in complex microbial communities, belonged among core microbiota families. Understanding the roles of individual population members in the total metabolism of the complex community may allow for interventions which might result in the replacement of Campylobacteraceae with Desulfovibrionaceae and a reduction of Campylobacter colonisation in broilers, carcasses, and consequently poultry meat products.

European validation of Real-Time PCR method for detection of Salmonella spp. in pork meat

The classical microbiological method for detection of Salmonella spp. requires more than five days for final confirmation, and consequently there is a need for an alternative methodology for detection of this pathogen particularly in those food categories with a short shelf-life. This study presents an international (at European level) ISO 16140-based validation study of a non-proprietary Real-Time PCR-based method that can generate final results the day following sample analysis. It is based on an ISO compatible enrichment coupled to an easy and inexpensive DNA extraction and a consolidated Real-Time PCR assay. Thirteen laboratories from seven European Countries participated to this trial, and pork meat was selected as food model. The limit of detection observed was down to 10 CFU per 25 g of sample, showing excellent concordance and accordance values between samples and laboratories (100%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (100%) when the results obtained for the Real-Time PCR-based methods were compared to those of the ISO 6579:2002 standard method. The results of this international trial demonstrate that the evaluated Real-Time PCR-based method represents an excellent alternative to the ISO standard. In fact, it shows an equal and solid performance as well as it reduces dramatically the extent of the analytical process, and can be easily implemented routinely by the Competent Authorities and Food Industry laboratories.

Epidemiological investigation of Chlamydophila psittaci in pigeons and free-living birds in Croatia.

During 2003, 278 adult pigeons (Columba livia) and 54 birds of 11 other free-living species were caught in the various locations in the City of Zagreb, Croatia. Sera from 182 pigeons were tested for the presence of antibodies against Chlamydophila (C.) psittaci by ELISA test and 174 of them (95.6%) were found positive. Because of the high positivity rate in sera, cloacal swabs of 278 pigeons as well as 54 other species of free-living birds were tested for the presence of C. psittaci antigen. Fourty-four of the 278 pigeons (15.83%) were antigen positive, whereas all 54 of the wild birds were negative. Antigen-positive pigeons were euthanised and examined pathomorphologically and cytologically. Findings of specific antibodies and antigen of C. psittaci confirmed the high rate of infection among urban pigeons in the City of Zagreb, fortunately not among other free-living birds. Although the pigeon serovars of C. psittaci are considered to be of moderate pathogenicity for humans, the identification of 15.8% antigen-positive birds represents a potential source of infection to humans, especially for elderly people and immunodeficient patients, as well as for poultry in the Zagreb city area.

Genetic analysis of Newcastle disease virus strains isolated in Bosnia-Herzegovina, Croatia, Slovenia and Yugoslavia, reveals the presence of only a single genotype, V, between 1979 and 2002.

Newcastle disease (ND) epizootics in some European countries after the World War II were caused by ND virus (NDV) of multiple genotypes (IV-VIIa) occurring sequentially and/or simultaneously. This study was carried out to characterise the genetic composition of NDV strains during the outbreaks in the territory of the former Yugoslavia in order to enhance our understanding of the relationships of past epizootics in Europe. Sixty-eight NDV strains isolated between 1979 and 2002 were analysed by restriction enzyme digestion and partial sequencing of the fusion protein gene. All isolates were placed in genotype V, an exotic type, that was introduced to western Europe in 1970. Residue substitution analysis has allowed the recognition of four genetic variants, Vb1-Vb4, and the tracing of their movements. Vb1, a dominant variant in Bulgaria from the late 1970s, was also wide spread in the former Yugoslavia throughout the period under investigation. Vb2, a variant occurring in the neighbouring countries in the early 1970s could be the founder of the epidemic in Yugoslavia and it was present up to the late 1980s. Variants Vb3 and Vb4 could be found only after 1987. In conclusion, the ND outbreaks in Yugoslavia were part of the epizootic wave due to genotype V viruses that started in western Europe in 1970 and became endemic in the region. Inter-country transmission occurred for all variants, and Vb3 and Vb4 might have evolved during the endemic period.

Microbes versus microbes: control of pathogens in the food chain.

Foodborne illness continues as a considerable threat to public health. Despite improved hygiene management systems and increased regulation, pathogenic bacteria still contaminate food, causing sporadic cases of illness and disease outbreaks worldwide. For many centuries, microbial antagonism has been used in food processing to improve food safety. An understanding of the mode of action of this microbial antagonism has been gained in recent years and potential applications in food and feed safety are now being explored. This review focuses on the potential opportunities presented, and the limitations, of using microbial antagonism as a biocontrol mechanism to reduce contamination along the food chain; including animal feed as its first link.

Isolation and molecular biological investigations of avian poxviruses from chickens, a turkey, and a pigeon in Croatia.

Abstract In the last 3 yr, several outbreaks of avian poxviruses (APVs) have been observed in different parts of Croatia. Four strains of APVs, from chickens, a pigeon, and a turkey, were isolated from cutaneous lesions by inoculation onto the chorioallantoic membranes (CAM) of 12-day-old specific-pathogen-free chicken embryos. The resulting proliferative CAM lesions contained eosinophilic cytoplasmic inclusion bodies. The characteristic viral particles of poxvirus were detected in the infected CAM and also in the infected tissues by transmission electron microscopy. Further identification and differentiation of the four various APVs were carried out by the use of a polymerase chain reaction (PCR) combined with restriction enzyme analysis. Using one primer set, which framed a region within the APV 4b core protein gene, it was possible to detect APV-specific DNA from all four tested isolates. PCR results revealed no recognizable differences in size of amplified fragments between the different APVs from chickens, turkey, and pigeon. Restriction enzyme analysis of PCR products using NlaIII showed the same cleavage pattern for turkey and chicken isolates and a different one for the pigeon isolate. Multiplex PCR for direct detection of APV and reticuloendotheliosis virus (REV) was carried out to determine the possible integration of REV in the genome of isolated APVs. The obtained results revealed that REV was present in chicken and turkey strains of poxviruses, whereas the pigeon isolate was negative. It is not known whether the avipoxvirus vaccine strain used in Croatia is contaminated with REV or if the REV is naturally contaminating Croatian field strains of fowl poxvirus. The latter is indicated by the negative REV finding in the pigeon, which was not vaccinated. The results of the present study indicate the reemergence of fowlpox in Croatia, where infections have not been recorded since 1963 and never confirmed etiologically.

Rapid Methods for Quality Assurance of Foods: the Next Decade with Polymerase Chain Reaction (PCR)-Based Food Monitoring

Microbiological analysis is an integral part of food quality control, as well as of the management of food chain safety. Microbiological testing of foodstuffs complements the preventive approach to food safety activities based mainly on implementation and application of the concept of Hazard Analysis and Critical Control Points (HACCP). Traditional microbiological methods are powerful but lengthy and cumbersome and therefore not fully compatible with current requirements. Even more, pathogens exist that are fastidious to cultivate or uncultivable at all. Besides immunological tests, molecular methods, specifically those based on polymerase chain reaction (PCR), are available options to meet industry and enforcement needs. The clear advantage of PCR over all other rapid methods is the striking analytical principle that is based on amplification of DNA, a molecule being present in every cell prone to multiply. Just by changing primers and probes, different genomes such as bacteria, viruses or parasites can be detected. A second advantage is the ability to both detect and quantify a biotic contaminant. Some previously identified obstacles of implementation of molecular methods have already been overcome. Technical measures became available that improved robustness of molecular methods, and equipment and biochemicals became much more affordable. Unfortunately, molecular methods suffer from certain drawbacks that hamper their full integration to food safety control. Those encompass a suitable sample pre-treatment especially for a quantitative extraction of bacteria and viruses from solid foods, limited availability of appropriate controls to evaluate the effectiveness of the analytical procedure, the current inability of molecular methods to distinguish DNA from viable cells and DNA from dead or non-cultivable cells, and the slow progress of international harmonisation and standardisation, which limit full acceptance of PCR-based methods in food control. The aim of this review is to describe the context and the prospects of PCR-based methods, as well as trends in research and development aimed at solving the next decade challenges in order to achieve full integration of molecular methods in food safety control.

PREVALENCE OF SALMONELLA IN CAPTIVE REPTILES FROM CROATIA

Salmonellosis transmitted by pet reptiles is an increasing public health issue worldwide. The aim of this study was to investigate the prevalence of Salmonella strains from captive reptiles in Croatia. From November 2009 to November 2011 a total of 292 skin, pharyngeal, cloacal, and fecal samples from 200 apparently healthy reptiles were tested for Salmonella excretions by bacteriologic culture and serotyping. These 200 individual reptiles included 31 lizards, 79 chelonians, and 90 snakes belonging to private owners or housed at the Zagreb Zoo, Croatia. Salmonella was detected in a total of 13% of the animals, among them 48.4% lizards, 8.9% snakes, and 3.8% turtles. Representatives of five of the six Salmonella enterica subspecies were identified with the following proportions in the total number of isolates: Salmonella enterica enterica 34.6%, Salmonella enterica houtenae 23.1%, Salmonella enterica arizonae 23.1%, Salmonella enterica diarizonae 15.4%, and Salmonella enterica salamae 3.8%. The 14 different serovars isolated included several rarely occurring serovars such as Salmonella Apapa, Salmonella Halle, Salmonella Kisarawe, and Salmonella Potengi. These findings confirm that the prevalence of Salmonella is considerable in captive reptiles in Croatia, indicating that these animals may harbor serovars not commonly seen in veterinary or human microbiologic practice. This should be addressed in the prevention and diagnostics of human reptile-transmitted infections.