Turkey adenovirus 3: ORF1 gene sequence comparison between vaccine-like and field strains.

Haemorrhagic enteritis is an economically significant disease reported in the majority of the countries where turkeys are raised intensively; it is caused by Turkey adenovirus 3 (TAdV-3). The aim of this study was to analyse and compare the ORF1 gene 3' region from turkey haemorrhagic enteritis virus (THEV) vaccine-like and field strains in order to develop a molecular diagnostic method to differentiate the strains from each other. Eighty samples were analysed by sequencing and phylogenetic analyses using a new set of polymerase chain reaction (PCR) primers targeting a genomic region spanning the partial ORF1, hyd and partial IVa2 gene sequences. A commercial live vaccine was also included in the analysis. The results showed that 56 of the 80 sequences obtained in this study showed ≥99.8% nucleotide identity with the homologous vaccine strain sequence. Three non-synonymous mutations - ntA1274G (aaI425V), ntA1420C (aaQ473H) and ntG1485A (aaR495Q) - were detected in the THEV field strains but not in the vaccine strain. Phylogenetic analysis confirmed the clustering of the field and vaccine-like strains in different phylogenetic branches. In conclusion, the method employed in this study could be a useful tool towards making a correct diagnosis. The data could contribute to the knowledge of field distribution of THEV strains and increase the limited existing information available on native isolates around the world.

Colistin Resistance Genes in Broiler Chickens in Tunisia.

Colistin is a polymyxin antibiotic that has been used in veterinary medicine for decades, as a treatment for enterobacterial digestive infections as well as a prophylactic treatment and growth promoter in livestock animals, leading to the emergence and spread of colistin-resistant Gram-negative bacteria and to a great public health concern, considering that colistin is one of the last-resort antibiotics against multidrug-resistant deadly infections in clinical practice. Previous studies performed on livestock animals in Tunisia using culture-dependent methods highlighted the presence of colistin-resistant Gram-negative bacteria. In the present survey, DNA extracted from cloacal swabs from 195 broiler chickens from six farms in Tunisia was tested via molecular methods for the ten mobilized colistin resistance (mcr) genes known so far. Of the 195 animals tested, 81 (41.5%) were mcr-1 positive. All the farms tested were positive, with a prevalence ranging from 13% to 93%. These results confirm the spread of colistin resistance in livestock animals in Tunisia and suggest that the investigation of antibiotic resistance genes by culture-independent methods could be a useful means of conducting epidemiological studies on the spread of antimicrobial resistance.

Marek's disease in genetically susceptible Cochin chickens in Italy: a case report.

The present study investigates an outbreak of classical Marek's disease (MD) in backyard Cochin chickens reared for hobby in Italy. Examined chickens showed spastic paralysis of the legs and at necropsy, enlargement and discoloration of the peripheral nerves and plexuses that matched microscopic A­ and B­ type MD lesions. Molecular analysis of the meq gene of the detected Gallid alphaherpesvirus 2 (GaHV­2) strain, showed typical markers of low virulence and the strain shared the entire meq gene sequence with strains circulating in Italian backyard chickens. Furthermore, the haplotype B19 of the major histocompatibility complex (MHC) was defined in the affected chickens, showing that the birds possessed a genetic profile of high susceptibility to MD, allowing the appearance of a classical nervous clinical form after infection with an apparently low pathogenicity GaHV­2 strain. Trade of live ornamental purebred chickens occurs frequently between hobby farmers and biosecurity practices, such as quarantine periods, should be applied to avoid the introduction of infected animals. Veterinarians should raise awareness of this issue and promote the use of vaccines against MD.

Occurrence of Chlamydiae in Corvids in Northeast Italy.

Chlamydiaceae occurrence has been largely evaluated in wildlife, showing that wild birds are efficient reservoirs for avian chlamydiosis. In this study, DNA extracted from cloacal swabs of 108 corvids from Northeast Italy was screened for Chlamydiaceae by 23S real-time (rt)PCR. The positive samples were characterised by specific rtPCRs for Chlamydia psittaci, Chlamydia abortus, Chlamydia gallinacea, Chlamydia avium, Chlamydia pecorum and Chlamydia suis. Cloacal shedding of Chlamydiaceae was detected in 12 out of 108 (11.1%, 5.9%-18.6% 95% CI) corvids sampled. Molecular characterisation at the species level was possible in 8/12 samples, showing C. psittaci positivity in only one sample from a hooded crow and C. abortus positivity in seven samples, two from Eurasian magpies and five from hooded crows. Genotyping of the C. psittaci-positive sample was undertaken via PCR/high-resolution melting, clustering it in group III_pigeon, corresponding to the B genotype based on former ompA analysis. For C. abortus genotyping, multilocus sequence typing was successfully performed on the two samples with high DNA load from Eurasian magpies, highlighting 100% identity with the recently reported Polish avian C. abortus genotype 1V strain 15-58d44. To confirm the intermediate characteristics between C. psittaci and C. abortus, both samples, as well as two samples from hooded crows, showed the chlamydial plasmid inherent in most C. psittaci and avian C. abortus, but not in ruminant C. abortus strains. The plasmid sequences were highly similar (≥99%) to those of the Polish avian C. abortus genotype 1V strain 15-58d44. To our knowledge, this is the first report of avian C. abortus strains in Italy, specifically genotype 1V, confirming that they are actively circulating in corvids in the Italian region tested.

Tetracycline Resistance Genes in Wild Birds from a Wildlife Recovery Centre in Central Italy.

Wild animals are less likely to be exposed directly to clinical antimicrobial agents than domestic animals or humans, but they can acquire antimicrobial-resistant bacteria through contact with humans, animals, and the environment. In the present study, 254 dead free-living birds belonging to 23 bird species were examined by PCR for the presence of tetracycline resistance (tet) genes. A fragment of the spleen was collected from each bird carcass. A portion of the intestine was also taken from 73 of the 254 carcasses. Extracted DNA was subjected to PCR amplification targeting the tet(L), tet(M), and tet(X) genes. In total, 114 (45%) of the 254 birds sampled belonging to 17 (74%) of the 23 bird species tested were positive for one or more tet genes. The tet(M) gene showed a higher frequency than the other tested genes, both in the spleen and in the intestine samples. These results confirm the potential role of wild birds as reservoirs, dispersers, or bioindicators of antimicrobial resistance in the environment.

Chlamydia suis and tetracycline resistance genes un Italian wild boar (Sus scrofa).

The aim of this study was to investigate the occurrence of Chlamydia suis and tetracycline resistance determinants in conjunctival swabs of Italian wild boars, by PCR. Extracted DNA collected from 50 wild boars from Northern and Central Italy was examined by molecular methods. One sample (2%) from the Central Italy was positive for C. suis. Fragments of tetR(C) and tetR(C)-tet(C) resistance determinants were amplified from the same sample. Further molecular investigations suggested the attribution of these tetracycline resistance determinants to C. suis, such as the truncation oftetR(C) and absence of a intact invasion (inv)-like region. While tetracycline-resistant C. suis is very common in domestic pigs, its occurrence has not been reported in wild boar before. Wild boar might acquire tetracycline resistance determinants through direct or indirect contact with domestic pigs.

High Frequency and Diversity of Tetracycline Resistance Genes in the Microbiota of Broiler Chickens in Tunisia.

Tetracycline resistance is still considered one of the most abundant antibiotic resistances among pathogenic and commensal microorganisms. The aim of this study was to evaluate the prevalence of tetracycline resistance (tet) genes in broiler chickens in Tunisia, and this was done by PCR. Individual cloacal swabs from 195 broiler chickens were collected at two slaughterhouses in the governorate of Ben Arous (Grand Tunis, Tunisia). Chickens were from 7 farms and belonged to 13 lots consisting of 15 animals randomly selected. DNA was extracted and tested for 14 tet genes. All the lots examined were positive for at least 9 tet genes, with an average number of 11 tet genes per lot. Of the 195 animals tested, 194 (99%) were positive for one or more tet genes. Tet(L), tet(M) and tet(O) genes were found in 98% of the samples, followed by tet(A) in 90.2%, tet(K) in 88.7% and tet(Q) in 80%. These results confirm the antimicrobial resistance impact in the Tunisian poultry sector and suggest the urgent need to establish a robust national antimicrobial resistance monitoring plan. Furthermore, the molecular detection of antibiotic resistance genes directly in biological samples seems to be a useful means for epidemiological investigations of the spread of resistance determinants.

Occurrence of Chicken Infectious Anemia Virus in Industrial and Backyard Tunisian Broilers: Preliminary Results.

Chicken infectious anemia virus (CIAV) is an economically important and widely distributed immunosuppressive agent in chickens. This study performed an epidemiological investigation on CIAV circulation in 195 Tunisian broilers, belonging to 13 lots from five industrial farms and in one rural farm. Fifteen animals were detected positive by a VP1 nested PCR. The amplicons were molecularly characterised by complete genome sequencing. All positive samples obtained in this study were from the rural farm, whereas the industrial farms sampled were negative. Nucleotide and amino acid sequence analyses showed a high degree of similarity among the sequences obtained, suggesting the circulation of a single CIAV strain in the positive lot. Phylogenetic analysis based on the CIAV VP1 nucleotide sequence and/or the complete genome showed that the sequences obtained in this study clustered with CIAV strains previously detected in Tunisia, Italy and Egypt, belonging to genogroup II. Our results highlight the need for constant CIAV surveillance in backyard chicken production.

PREVALENCE OF CHLAMYDIACEAE AND TETRACYCLINE RESISTANCE GENES IN WILD BOARS OF CENTRAL EUROPE.

Our aim was to investigate the occurrence and distribution of Chlamydia suis and other Chlamydiaceae in the wild boar (Sus scrofa) population of Switzerland and Northern Italy and the detection of tetracycline resistance genes by PCR. We collected a total of 471 conjunctival swabs (n=292), rectal swabs (n=147), and lung tissue samples (n=32) belonging to 292 wild boars. The prevalence of Chlamydiaceae in the investigated wild boar populations was very low (1.4%, 4/292). We found C. suis in rectal or conjunctival swabs but not in lung samples. The low chlamydial prevalence might be attributed to limited contacts between wild boars and outdoor domestic pigs due to strict biosecurity measures or limited numbers of rural pig herds. The tetA(C) gene fragment was detected in six samples, which were all negative for Chlamydiaceae, and was probably not of chlamydial origin but more likely from other bacteria. The low tetracycline resistance rate in wild boar might be explained by the lack of selective pressure. However, transmission of resistance genes from domestic pigs to wild boar or selective pressure in the environment could lead to the development and spread of tetracycline-resistant C. suis strains in wild boars.

Molecular detection and characterization of Mycoplasma gallisepticum and Mycoplasma synoviae strains in backyard poultry in Italy.

Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) represent the most important avian Mycoplasma species in the poultry industry, causing considerable economic losses. In Italy, the presence of MG or MS has been investigated especially in commercial poultry farms. To our knowledge, no systematic investigations on MG or MS presence using highly specific diagnostic assays have been performed in backyard poultry. The aim of this study was to detect and molecularly characterize MG and MS strains in 11 backyard poultry flocks located in different regions of Italy. Tracheal swabs were collected and DNA was extracted. For MS, a PCR targeting a vlhA gene fragment was performed, and typing and subtyping was attempted. The presence of MG was investigated by a screening PCR, then MG typing by gene-targeted sequencing (GTS). All the amplicons were sequenced, then MG and MS dendrograms were constructed. All the flocks examined resulted Mycoplasma positive: 5 out of 11 (45.45%) were MG and MS positive, 3 (27.27%) were MG positive, and the remaining 3 (27.27%) were MS positive. The MS detections were assigned to types C, D, and F. All strains of type D belonged to subtype D1 and 2 unknown subtypes were identified. A MS sequence showed peculiar characteristics, which did not allow assignment to a known MS type or subtype. MG GTS analysis identified 6 MG strains belonging to 5 subclusters circulating in Italian backyards chicken flocks. The results of this study provide evidence of a risk for commercial poultry farms, especially in areas where backyard and commercial farms are close, suggesting the implementation of biosecurity measures.

Detection of Tetracycline Resistance Genes in European Hedgehogs (Erinaceus europaeus) and Crested Porcupines (Hystrix cristata).

Relatively little is known regarding the role of wildlife in the development of antibiotic resistance. Our aim was to assess the presence of the tetracycline resistance genes, tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(K), tet(L), tet(M), tet(O), tet(P), tet(Q), tet(S), and tet(X), in tissue samples of 14 hedgehogs (Erinaceus europaeus) and 15 crested porcupines (Hystrix cristata) using PCR assays. One or more tet genes were found in all but three hedgehogs and one crested porcupine. Of the 14 tetracycline resistance genes investigated, 13 were found in at least one sample; tet(G) was not detected. We confirmed the potential role of wild animals as bioindicators, reservoirs, or vectors of antibiotic-resistant bacteria in the environment.

Chlamydiosis in Backyard Chickens (Gallus gallus) in Italy.

Until recently, Chlamydia psittaci was considered to be the only etiological agent of avian chlamydiosis, but two new avian species, Chlamydia gallinacea and Chlamydia avium, have recently been described in poultry and pigeons or psittacine birds, respectively. The aim of this study was to explore the occurrence of C. psittaci and C. gallinacea in backyard chickens in Italy. Cloacal swabs were taken from 160 asymptomatic chickens reared in 16 backyard farms. Samples were tested for C. psittaci and C. gallinacea by specific real-time polymerase chain reaction assays, with 24 (15%) of the 160 chickens resulting positive for C. gallinacea. To attempt chlamydial isolation, new samples were obtained from two farms harboring a high prevalence (60% and 70%, respectively) of C. gallinacea-positive chickens. In total, eight C. gallinacea and one C. psittaci isolates were successfully recovered from 13 chickens. C. gallinacea was confirmed to be the endemic chlamydial species in chickens, with a high ompA intraspecies diversity. The presence of viable C. psittaci and C. gallinacea demonstrated by isolation from chickens in backyard farms poses a potential public health problem.

Distinct Leishmania infantum Strains Circulate in Humans and Dogs in the Emilia-Romagna Region, Northeastern Italy.

Human leishmaniasis is an emerging problem in Italy and is on the increase in the Emilia-Romagna region, northeastern part of the country. Nevertheless, studies dealing with the molecular characterization of Leishmania spp. circulating in these areas are limited. In the present work, we explored the genetic polymorphism of Leishmania isolates from 28 cases of canine leishmaniasis and three cases of human visceral leishmaniasis (VL), which occurred in 2013-2014 in the Emilia-Romagna region. The characterization was carried out in comparison with nine human isolates of Leishmania from other VL endemic Italian regions and two reference strains. Nucleic acid from 31 Leishmania-positive phlebotomine sandfly pools, sampled in 2012-2013 in the Emilia-Romagna region, were also evaluated. DNA amplification and sequencing of the ribosomal internal transcribed spacer-1 and of a repetitive nuclear region on chromosome 31 were carried out for genotyping. Two size polymorphic targets were also analyzed by PCR, the cpb E/F-gene and the k26-gene. Altogether, the analysis showed the circulation of different Leishmania infantum genotypes in the Emilia-Romagna region: two genotypes found in dogs from public kennels were similar to VL isolates from other Italian regions, whereas a third genotype was detected in VL cases of the Emilia-Romagna region and in all but one of the sandfly pools. The combined molecular tools applied in this study can constitute a helpful support for parasite tracking (e.g., in outbreak investigations) and for a better understanding of the epidemiological evolution of leishmaniasis in northeastern Italy.

The Chlamydia suis Genome Exhibits High Levels of Diversity, Plasticity, and Mobile Antibiotic Resistance: Comparative Genomics of a Recent Livestock Cohort Shows Influence of Treatment Regimes.

Chlamydia suis is an endemic pig pathogen, belonging to a fascinating genus of obligate intracellular pathogens. Of particular interest, this is the only chlamydial species to have naturally acquired genes encoding for tetracycline resistance. To date, the distribution and mobility of the Tet-island are not well understood. Our study focused on whole genome sequencing of 29 C. suis isolates from a recent porcine cohort within Switzerland, combined with data from USA tetracycline-resistant isolates. Our findings show that the genome of C. suis is very plastic, with unprecedented diversity, highly affected by recombination and plasmid exchange. A large diversity of isolates circulates within Europe, even within individual Swiss farms, suggesting that C. suis originated around Europe. New World isolates have more restricted diversity and appear to derive from European isolates, indicating that historical strain transfers to the United States have occurred. The architecture of the Tet-island is variable, but the tetA(C) gene is always intact, and recombination has been a major factor in its transmission within C. suis. Selective pressure from tetracycline use within pigs leads to a higher number of Tet-island carrying isolates, which appear to be lost in the absence of such pressure, whereas the loss or gain of the Tet-island from individual strains is not observed. The Tet-island appears to be a recent import into the genome of C. suis, with a possible American origin.

Selective Pressure Promotes Tetracycline Resistance of Chlamydia Suis in Fattening Pigs.

In pigs, Chlamydia suis has been associated with respiratory disease, diarrhea and conjunctivitis, but there is a high rate of inapparent C. suis infection found in the gastrointestinal tract of pigs. Tetracycline resistance in C. suis has been described in the USA, Italy, Switzerland, Belgium, Cyprus and Israel. Tetracyclines are commonly used in pig production due to their broad-spectrum activity and relatively low cost. The aim of this study was to isolate clinical C. suis samples in cell culture and to evaluate their antibiotic susceptibility in vitro under consideration of antibiotic treatment on herd level. Swab samples (n = 158) identified as C. suis originating from 24 farms were further processed for isolation, which was successful in 71% of attempts with a significantly higher success rate from fecal swabs compared to conjunctival swabs. The farms were divided into three treatment groups: A) farms without antibiotic treatment, B) farms with prophylactic oral antibiotic treatment of the whole herd consisting of trimethoprime, sulfadimidin and sulfathiazole (TSS), or C) farms giving herd treatment with chlortetracycline with or without tylosin and sulfadimidin (CTS). 59 isolates and their corresponding clinical samples were selected and tested for the presence or absence of the tetracycline resistance class C gene [tet(C)] by conventional PCR and isolates were further investigated for their antibiotic susceptibility in vitro. The phenotype of the investigated isolates was either classified as tetracycline sensitive (Minimum inhibitory concentration [MIC] < 2 µg/ml), intermediate (2 µg/ml ≤ MIC < 4 µg/ml) or resistant (MIC ≥ 4 µg/ml). Results of groups and individual pigs were correlated with antibiotic treatment and time of sampling (beginning/end of the fattening period). We found clear evidence for selective pressure as absence of antibiotics led to isolation of only tetracycline sensitive or intermediate strains whereas tetracycline treatment resulted in a greater number of tetracycline resistant isolates.

Molecular characterization of Leishmania infantum strains by kinetoplast DNA RFLP-PCR.

Multilocus enzyme electrophoresis is the tool most frequently used to classify Leishmania spp., although it is time consuming and, sometimes, a not enough discriminative method. In the present study a kinetoplast DNA polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to characterize 16 zymodeme MON-1 Leishmania infantum strains: 15 were from dogs housed in public kennels of 7 geographical areas in the Emilia-Romagna region, Northern Italy, 1 was the L. infantum reference strain MHOM/TN/1980/IPT1. Six enzymatic patterns were observed. Kinetoplast DNA RFLP-PCR confirmed to have a good discriminatory power within the same zymodeme and proved to be useful for comparing few strains or discriminating between relapse and reinfection in the same host. We therefore recommend it use for discriminating between relapse and reinfection in the same host rather than supporting large-scale epidemiological studies.

Tetracycline Susceptibility in Chlamydia suis Pig Isolates.

The aims of the present study were to assess the prevalence of Chlamydia suis in an Italian pig herd, determine the tetracycline susceptibility of C. suis isolates, and evaluate tet(C) and tetR(C) gene expression. Conjunctival swabs from 20 pigs were tested for C. suis by real-time polymerase chain reaction, and 55% (11) were positive. C. suis was then isolated from 11 conjunctival swabs resampled from the same herd. All positive samples and isolates were positive for the tet(C) resistance gene. The in vitro susceptibility to tetracycline of the C. suis isolates showed MIC values ranging from 0.5 to 4 µg/mL. Tet(C) and tetR(C) transcripts were found in all the isolates, cultured both in the absence and presence of tetracycline. This contrasts with other Gram-negative bacteria in which both genes are repressed in the absence of the drug. Further investigation into tet gene regulation in C. suis is needed.

Prevalence of Chlamydial Infections in Fattening Pigs and Their Influencing Factors.

Chlamydial infections in pigs are associated with respiratory disease, diarrhea, conjunctivitis and other pathologies. The aim of this study was to define the prevalence of Chlamydiaceae in Swiss fattening pigs by applying sensitive and specific detection methods and to correlate prior antibiotic treatment and farm related factors with differences in prevalence. Conjunctival and fecal swabs were collected from 636 pigs in 29 Swiss fattening pig farms with and without antibiotic treatment, at the beginning and the end of the fattening period. The swabs were screened by real-time PCR for Chlamydiaceae. For the chlamydial detection and species-identification, a DNA-microarray analysis was performed. All farms were positive for Chlamydiaceae with 94.3 and 92.0% prevalence in fecal swabs as well as 45.9 and 32.6% in conjunctival swabs at the first and second time points, respectively. Antibiotic treatment could not clear the infection on herd level. Potential contact with wild boars was a significant risk factor, while hygiene criteria did not influence chlamydial prevalence. A correlation of chlamydial positivity to diarrhea, but not to conjunctivitis was evident. Chlamydia suis was the predominant species. Mixed infections with C. suis and C. pecorum were common, with a substantial increase in C. pecorum positivity at the end of the fattening period, and this finding was associated with ruminant contact. C. abortus was detected in one conjunctival swab. In this study, C. suis inhabited the intestinal tract of nearly all examined pigs, implying a long-term infection. C. pecorum was also common and might be transmitted to pigs by ruminants.

Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii Circulation in a Dairy Farm and Sources of Milk Contamination.

Even though dairy cows are known carriers of Arcobacter species and raw or minimally processed foods are recognized as the main sources of human Arcobacter infections in industrialized countries, data on Arcobacter excretion patterns in cows and in milk are scant. This study aimed to identify potentially pathogenic Arcobacter species in a dairy herd and to investigate the routes of Arcobacter transmission among animals and the potential sources of cattle infection and milk contamination. A strategy of sampling the same 50 dairy animals, feed, water, and milk every month for a 10-month period, as well as the sampling of quarter milk, animal teats, the milking environment, and animals living on the farm (pigeons and cats), was used to evaluate, by pulsed-field gel electrophoresis (PFGE), the characteristic patterns in animals, their living environment, and the raw milk they produced. Of the 463 samples collected, 105 (22.6%) were positive for Arcobacter spp. by culture examination. All the matrices except quarter milk and pigeon gut samples were positive, with prevalences ranging from 15 to 83% depending on the sample. Only three Arcobacter species, Arcobacter cryaerophilus (54.2%), A. butzleri (34.2%), and A. skirrowii (32.3%), were detected. PFGE analysis of 370 isolates from positive samples provided strong evidence of Arcobacter circulation in the herd: cattle likely acquire the microorganisms by orofecal transmission, either by direct contact or from the environment, or both. Water appears to be a major source of animal infection. Raw milk produced by the farm and collected from a bulk tank was frequently contaminated (80%) by A. butzleri; our PFGE findings excluded primary contamination of milk, whereas teats and milking machine surfaces could be sources of Arcobacter milk contamination.

A mouse model for Chlamydia suis genital infection.

A mouse model for Chlamydia suis genital infection was developed. Ninety-nine mice were randomly divided into three groups and intravaginally inoculated with chlamydia: 45 mice (group 1) received C. suis purified elementary bodies (EBs), 27 (group 2) were inoculated with C. trachomatis genotype E EBs and 27 mice (group 3) with C. trachomatis genotype F EBs. Additionally, 10 mice were used as a negative control. At seven days post-infection (dpi) secretory anti-C. suis IgA were recovered from vaginal swabs of all C. suis inoculated mice. Chlamydia suis was isolated from 93, 84, 71 and 33% vaginal swabs at 3, 5, 7 and 12 dpi. Chlamydia trachomatis genotype E and F were isolated from 100% vaginal swabs up to 7 dpi and from 61 and 72%, respectively, at 12 dpi. Viable C. suis and C. trachomatis organisms were isolated from uterus and tubes up to 16 and 28 dpi, respectively. The results of the present study show the susceptibility of mice to intravaginal inoculation with C. suis. A more rapid course and resolution of C. suis infection, in comparison to C. trachomatis, was highlighted. The mouse model could be useful for comparative investigations involving C. suis and C. trachomatis species.