An improved multiplex PCR for Actinobacillus pleuropneumoniae, Glaesserella australis and Pasteurella multocida.

Glaesserella australis, a newly described bacterial species, has been isolated from pig lungs that displayed lesions very similar to those caused by Actinobacillus pleuropneumoniae, prompting the need for a validated diagnostic tool. In this work, we have altered a multiplex PCR used for the identification of cultures of G. australis, A. pleuropneumoniae and Pasteurella multocida to be more sensitive and then evaluated the use of the altered diagnostic tool on cultures and directly on tissues. The altered multiplex PCR was validated using 47 related species, both type/reference strains and field isolates. The sensitivity was assessed by serial dilutions and used a mixture of target bacteria in different concentrations. Further, 166 lung samples from 54 farms from four Australian States were used to validate the ability of the multiplex PCR to detect bacteria in lung swabs. The multiplex PCR was specific for the three target species. The assay could detect a minimum of 40 colony forming units (CFU) of G. australis, 786 CFU of A. pleuropneumoniae and 238 CFU of P. multocida. The multiplex PCR yielded more positives than coventional bacteriological examination. From a total of 166 lung samples, 51.9%, 51.9% and 5.6% of farms were PCR positive for P. multocida, A. pleuropneumoniae and G. australis, respectively. The results suggested that the new multiplex PCR was specific, sensitive and out performed traditional culture. The prevalence of G. australis was not very high, but it was the dominant pathogen in infected pigs.

Clonal spread of multi-resistant Gallibacterium anatis isolates among Iranian broilers and layers.

Gallibacterium anatis is a common cause of reproductive tract infection in chickens, which leads to reduced egg production and increased mortality. This study was undertaken to investigate prevalence of G. anatis in 12 poultry flocks originating from Iranian provinces with leading chicken production and to determine genetic diversity, antimicrobial resistance, and the presence of major antigens of the isolates investigated. Out of the 120 chicken tracheal samples collected and tested, 84 (70%) were positive for G. anatis. Genotyping by Pulse Field Gel Electrophoresis and genome sequencing revealed a total of 24 pulsotypes for 71 strains (at a 87% similarity level) and seven genome clusters comprising 21 strains (97% similarity level), respectively. The combination of the two typing methods confirmed the presence of several genotypes originating from a common ancestor affecting poultry yet also suggested that identical clones were shared among chickens within farms and between different farms. The latter finding is to our knowledge the first example of clonal presence of G. anatis in epidemiologically unrelated farms. The 21 sequenced strains were characterized against a panel of commonly used antibiotics and showed lowered sensitivity to tetracycline (76.2%) and enrofloxacin (90.5%). The widespread presence of multiresistant G. anatis isolates calls for non-antibiotic prophylactics. Three major immunogen genes, gtxA, Gab_1309 and Gab_2312 were detected in the isolates indicating these antigens likely represent effective vaccine targets. A conserved sequence of the gtxA gene across a range of epidemiologically independent strains suggests the use of GtxA for future vaccine development purposes.

Occurrence of Pasteurellaceae and Neisseriaceae bacteria in the pharyngeal and respiratory tract of dogs and cats - Short communication.

The occurrence of members of the Pasteurellaceae and Neisseriaceae families was studied in dogs and cats. A total of 110 nasal and pharyngeal swab samples from 47 dogs and 8 cats were collected. Most of the strains were identified by 16S rDNA sequencing, except Frederiksenia canicola and Pasteurella multocida where species-specific polymerase chain reactions were applied. The most frequently isolated species was F. canicola, which occurred only in dogs, mainly in the pharyngeal cavity. The second commonest bacterium, P. multocida was found in both types of samples and in both hosts. Other species from the family Pasteurellaceae, such as Haemophilus haemoglobinophilus, Pasteurella canis and P. dagmatis, were detected only in dogs. All isolated species belonging to the family Neisseriaceae, mainly representing Neisseria weaveri, were found only in the pharyngeal cavity. Neisseria weaveri and N. zoodegmatis could be detected in both hosts. Neisseria dumasiana and N. canis were isolated from dogs, while N. shayeganii only from a cat. For phylogenetic analysis, rpoB gene sequencing was performed, where the strains were on monophyletic branches and clearly separated from each other. In this study, recently described species such as F. canicola, N. shayeganii and N. dumasiana were detected that had never been isolated in Hungary before.

Prevalence and antimicrobial resistance of opportunistic pathogens associated with bovine respiratory disease isolated from nasopharyngeal swabs of veal calves in Switzerland.

The composition of the bacterial flora in the calf nasopharynx might influence the risk of bovine respiratory disease (BRD). The aims of the present study were, firstly, to investigate the prevalence of bacteria potentially involved in BRD in the nasopharynx of veal calves and to identify associated risk factors for their presence, and, secondly, to provide data on antimicrobial resistance levels in these bacteria. Deep nasopharyngeal swabs were collected from veal calves on 12 Swiss farms over a period of one year by non-random, but systematic sampling for isolation of Pasteurellaceae and Mycoplasma (M.) bovis and dispar. Associations of potential risk factors with occurrence of these bacteria were tested in multivariable mixed logistic regression analyses, based on information gained from extensive questionnaires completed with the farmers. Antimicrobial susceptibility testing was performed for Pasteurellaceae by broth microdilution method to obtain minimal inhibitory concentrations (MIC). Pasteurellaceae, including Pasteurella (P.) multocida, Mannheimia (M.) haemolytica, Bisgaard Taxon 39 and Histophilus (H.) somni, were almost twice as prevalent as M. bovis and dispar in this study. Continuous stocking was a risk factor for the presence of Pasteurellaceae, especially when calves originated from more than six suppliers. In young calves (≤ 91 days), feeding of California Mastitis Test (CMT) positive milk was an additional risk factor for the presence of Pasteurellaceae whereas transport of calves by farmers and livestock traders (as opposed to transport only by farmers) increased the risk in older calves (> 91 days). Risk factors for the presence of M. bovis/dispar were higher number of calves per drinking nipple in young calves, and no access to an outside pen and feeding of CMT positive milk in older calves, respectively. While further research will have to investigate the observed associations in more detail, this suggests that management can play an important role in the prevalence of nasopharyngeal bacteria with a potential subsequent involvement in BRD. Antimicrobial resistance differed between the three bacterial species tested in this study and was highest to oxytetracycline and spectinomycin in P. multocida, oxytetracycline and penicillin in M. haemolytica, and ampicillin and penicillin in H. somni. Only two European VetCAST breakpoints (for florfenicol in P. multocida and M. haemolytica) have been published to date, matching the MIC distribution of the present isolate populations well, in contrast to certain commonly applied American Clinical and Laboratory Institute interpretive criteria. This highlights the potential for further refinement of clinical breakpoints in veterinary medicine.

How sure are you? A web-based application to confront imperfect detection of respiratory pathogens in bighorn sheep.

The relationships between host-pathogen population dynamics in wildlife are poorly understood. An impediment to progress in understanding these relationships is imperfect detection of diagnostic tests used to detect pathogens. If ignored, imperfect detection precludes accurate assessment of pathogen presence and prevalence, foundational parameters for deciphering host-pathogen dynamics and disease etiology. Respiratory disease in bighorn sheep (Ovis canadensis) is a significant impediment to their conservation and restoration, and effective management requires a better understanding of the structure of the pathogen communities. Our primary objective was to develop an easy-to-use and accessible web-based Shiny application that estimates the probability (with associated uncertainty) that a respiratory pathogen is present in a herd and its prevalence given imperfect detection. Our application combines the best-available information on the probabilities of detection for various respiratory pathogen diagnostic protocols with a hierarchical Bayesian model of pathogen prevalence. We demonstrated this application using four examples of diagnostic tests from three herds of bighorn sheep in Montana. For instance, one population with no detections of Mycoplasma ovipneumoniae (PCR assay) still had an 6% probability of the pathogen being present in the herd. Similarly, the apparent prevalence (0.32) of M. ovipneumoniae in another herd was a substantial underestimate of estimated true prevalence (0.46: 95% CI = [0.25, 0.71]). The negative bias of naïve prevalence increased as the probability of detection of testing protocols worsened such that the apparent prevalence of Mannheimia haemolytica (culture assay) in a herd (0.24) was less than one third that of estimated true prevalence (0.78: 95% CI = [0.43, 0.99]). We found a small difference in the estimates of the probability that Mannheimia spp. (culture assay) was present in one herd between the binomial sampling approach (0.24) and the hypergeometric approach (0.22). Ignoring the implications of imperfect detection and sampling variation for assessing pathogen communities in bighorn sheep can result in spurious inference on pathogen presence and prevalence, and potentially poorly informed management decisions. Our Shiny application makes the rigorous assessment of pathogen presence, prevalence and uncertainty straightforward, and we suggest it should be incorporated into a new paradigm of disease monitoring.

Prevalence and temporal trends in antimicrobial resistance of bovine respiratory disease pathogen isolates submitted to the Wisconsin Veterinary Diagnostic Laboratory: 2008-2017.

The objective of this study was to describe the prevalence and trends in antimicrobial resistance for bacterial pathogens associated with bovine respiratory disease (BRD) isolated from samples submitted to the Wisconsin Veterinary Diagnostic Laboratory (WVDL). Data were retrospectively collected from bovine respiratory isolates including Pasteurella multocida, Mannheimia haemolytica, Histophilus somni, and Bibersteinia trehalosi identified at the WVDL between January 2008 and December 2017. Antimicrobial susceptibility testing data were queried from antimicrobial resistance databases at the WVDL. A total of 4,261 isolates were identified. Pasteurella multocida was most frequently identified, accounting for 2,094 isolates (49% of total) over the study period. Mannheimia haemolytica was the second most frequently isolated bacterial respiratory pathogen (n = 1,267, 30%) followed by H. somni (n = 749, 18%) and B. trehalosi (n = 151, 4%). Over the 10-yr period, B. trehalosi had the highest median percentage of isolates that were resistant to at least one antibiotic at 33% (interquartile range: 24, 47) followed by M. haemolytica (13%; 8, 23). For P. multocida, 10% (4, 26) of isolates were classified as resistant to at least one antibiotic, whereas H. somni had the fewest resistant isolates (9%; 3, 15). When comparing 2013-2017 to 2008-2012, the overall percentage of resistant isolates for P. multocida and B. trehalosi decreased, whereas the percentage of resistant isolates for M. haemolytica and H. somni increased. Increased resistance against florfenicol, fluoroquinolones, gentamicin, tilmicosin, and tulathromycin was observed for M. haemolytica. These data show that antimicrobial susceptibility for BRD bacterial pathogens has changed in the population served by the WVDL over this 10-yr period. For P. multocida, resistance is relatively low and has either improved or at least remained constant for the majority of drugs labeled for treatment of respiratory disease in dairy cattle. Veterinarians and producers should be aware of the bacterial pathogens most commonly associated with BRD and work toward early disease detection, proper antibiotic administration, and monitoring lung lesions to ensure that their treatment protocols improve lung health.

High phylogenetic diversity of Gallibacterium anatis is correlated with low biosecurity measures and management practices on poultry farms.

Gallibacterium anatis is considered one of the most common bacterial causative agents of reproductive tract disorders in poultry. In this study, phylogenetic analysis of partial rpoB sequences and biotyping using MALDI-TOF MS was done in order to investigate the genetic diversity of Gallibacterium isolates from 13 farms with different biosecurity measures and management practices. Sampling was done as a part of regular monitoring, except for Farms 9-13 that were included in the study to represent extensive production systems with lowest biosecurity levels. Pharyngeal and cloacal swabs were taken from live birds, while swabs from trachea, liver, peritoneum and oviduct were taken during necropsies. After cultivation and identification, strains from each farm were randomly selected for sequencing and biotyping. Both results showed high level of heterogeneity among the isolates originating from farms with low biosecurity levels, unlike isolates from farms with higher biosecurity levels and proper management that were more closely related and clustered together. Such correlation was statistically significant. Low biosecurity levels enable horizontal transmission of the pathogens, as well as gene transfer. The results confirm the importance of adequate biosecurity measures and management on poultry farms as they greatly affect the genetic diversity of the pathogens. Therefore, implementation of basic biosecurity measures could help control the heterogeneity of Gallibacterium strains, which would alleviate control of the infection prevalence on farms through immunoprophylaxis, and consequently improve poultry production. Also, the genetic diversity of G. anatis on poultry farms could be a good bioindicator of management practices and biosecurity measures used. RESEARCH HIGHLIGHTS High correlation between low biosecurity and high diversity of Gallibacterium anatis. Diversity of Gallibacterium is a good bioindicator of management practices on farms.

Etiology, epidemiology, pathology, and advances in diagnosis, vaccine development, and treatment of Gallibacterium anatis infection in poultry: a review.

Gallibacterium anatis is a Gram-negative bacterium of the Pasteurellaceae family that resides normally in the respiratory and reproductive tracts in poultry. It is a major cause of oophoritis, salpingitis, and peritonitis, decreases egg production and mortality in hens thereby severely affecting animal welfare and overall productivity by poultry industries across Europe, Asia, America, and Africa. In addition, it has the ability to infect wider host range including domesticated and free-ranging avian hosts as well as mammalian hosts such as cattle, pigs and human. Evaluating the common virulence factors including outer membrane vesicles, fimbriae, capsule, metalloproteases, biofilm formation, hemagglutinin, and determining novel factors such as the RTX-like toxin GtxA, elongation factor-Tu, and clustered regularly interspaced short palindromic repeats (CRISPR) has pathobiological, diagnostic, prophylactic, and therapeutic significance. Treating this bacterial pathogen with traditional antimicrobial drugs is discouraged owing to the emergence of widespread multidrug resistance, whereas the efficacy of preventing this disease by classical vaccines is limited due to its antigenic diversity. It will be necessary to acquire in-depth knowledge on important virulence factors, pathogenesis and, concerns of rising antibiotic resistance, improvised treatment regimes, and novel vaccine candidates to effectively tackle this pathogen. This review substantially describes the etio-epidemiological aspects of G. anatis infection in poultry, and updates the recent development in understanding the pathogenesis, organism evolution and therapeutic and prophylactic approaches to counter G. anatis infection for safeguarding the welfare and health of poultry.

Genotypic divergence of Avibacterium paragallinarum isolates with different growth requirements for nicotinamide adenine dinucleotide.

In 2017, for the first time in Asia, we reported the isolation of variants of Avibacterium paragallinarum with atypical NAD dependency. The present study was conducted to characterize the genotypes of 24 isolates of Av. paragallinarum in Korea, including the four variants reported previously. Most of the typical isolates (19/20) showed a unique ERIC-PCR pattern with no ERIC-PCR patterns in common between the typical isolates and the variants. Furthermore, the variants shared no ERIC-PCR patterns among themselves. All the typical NAD-dependent isolates belonged to the same phylogenetic group based on both 16S rRNA and hagA gene sequences. The four variants were placed in several groups distinct from the typical isolates. In the 16S rRNA phylogenetic analysis, two of the variants were not closely aligned to any other Av. paragallinarum, isolate although they were clearly members of the genus Avibacterium. The other variants were clustered together with NAD atypical isolates from geographically diverse global locations. Compared with the Modesto reference strain AY498870, all the variants lacked a TTTTT stretch at positions 182-186 in the 16S rRNA gene and the same deletion was shown in most of the reported variants. The typical isolates and variants shared 97.3-98.2% and 95.2-97.2% nucleotide sequence similarity, for 16S rRNA and hagA, respectively. In addition, the similarities among variants were within 98.3-100% and 96.5-98.4% for the two genes, respectively. Our results indicate that the Av. paragallinarum variants with altered NAD growth requirements were genetically different and highly divergent from the typical NAD-dependent isolates.RESEARCH HIGHLIGHTS NAD variant Korean Av. paragallinarum isolates show genetic diversity, whereas typical Korean Av. paragallinarum isolates do not.The Korean variants were not closely aligned to all other Av. paragallinarum in the 16S rRNA phylogeny.NAD atypical isolates from geographically diverse global locations clustered together.Almost all variants, including all Korean variants of Av. paragallinarum, lack a specific fragment of the 16S rRNA gene.

First Report of Isolation of Gallibacterium anatis from Layer Chickens in Morocco with Decrease in Laying Performance.

Gallibacterium is a genus of the family of Pasteurellaceae. It is well known as a commensal inhabitant of the respiratory and reproductive tract of healthy chickens. But in the last years, Gallibacterium anatis is increasingly reported in field cases with a decrease in laying performance due to infections of the reproductive tract. The aim of the present study was to investigate the implication of G. anatis infection in layer flocks facing a decrease in laying performance in Morocco. Birds were received from five different laying hen farms in two regions in Morocco showing a drop of egg production. Necropsy revealed 46.1 % (24/52) of sampled birds showed variable lesions in ovaries, salpinx, and trachea. In fact, 24 birds were affected by salpingitis, 18 by oophoritis, and 11 birds by atrophy of ovaries. Furthermore, tracheitis was observed in 24 birds. Bacteriological investigation was done from different organs, and G. anatis was found in ovaries (n = 20), trachea (n = 17), and cloaca (n = 3). Identification was based on growth morphology, Gram staining, and biochemical properties. Additionally, polymerase chain reaction test using specific primers for the genus identification was carried out. All isolates showed bands of 925 bp specific for G. anatis expressing the virulent toxin GtxA. Antibiotic resistance testing was performed and revealed that isolates were sensitive to enrofloxacin, florfenicol, and gentamycin but resistant to ampicillin, erythromycin, oxytetracycline, and sulfamethoxazole-trimethoprim. The present study is the first report of G. anatis in Morocco, demonstrating the need for further epidemiologic investigations as well as in regard to antibiotic resistance development.

Primer informe de aislamiento de Gallibacterium anatis de gallinas de postura en Marruecos con disminución en la eficiencia de la postura. Gallibacterium es un género de la familia Pasteurellaceae. Es bien conocido como un habitante comensal del tracto respiratorio y reproductivo de pollos sanos. Pero en los últimos años, han aumentado los reportes de Gallibacterium anatis en casos de campo con una disminución en la eficiencia de la postura debido a infecciones del tracto reproductivo. El objetivo del presente estudio fue investigar la implicación de la infección por G. anatis en parvadas de gallinas de postura que presentaban una disminución en la eficiencia de la postura en Marruecos. Se recibieron aves de cinco granjas diferentes de gallinas postura en dos regiones de Marruecos, mostrando una caída en la producción de huevos. La necropsia reveló que 46.1% (24/52) de las aves muestreadas mostraron lesiones variables en ovarios, oviducto y tráquea. De hecho, 24 aves mostraron salpingitis, 18 ooforitis y 11 aves atrofia de ovarios. Además, se observó traqueítis en 24 aves. Se realizó la investigación bacteriológica de diferentes órganos, y se encontró G. anatis en ovarios (n = 20), tráquea (n = 17) y cloaca (n = 3). La identificación se basó en la morfología del crecimiento, en la tinción de Gram y en las propiedades bioquímicas. Además, se realizó una prueba de reacción en cadena de la polimerasa utilizando iniciadores específicos para la identificación del género. Todos los aislamientos mostraron bandas de 925 pares de bases específicas para G. anatis que expresaban la toxina virulenta GtxA. Se realizaron pruebas de resistencia a los antibióticos y revelaron que los aislamientos eran sensibles a enrofloxacina, florfenicol y gentamicina, pero resistentes a ampicilina, eritromicina, oxitetraciclina y sulfametoxazol-trimetoprima. El presente estudio es el primer informe de G. anatis en Marruecos, que demuestra la necesidad de más investigaciones epidemiológicas, así como en relación con el desarrollo de resistencia a los antibióticos.

Identification and characterization of Dutch Avibacterium paragallinarum isolates and the implications for diagnostics.

This study reports the results of diagnostic and molecular typing methods for 18 Avibacterium paragallinarum isolates obtained from outbreaks of infectious coryza in commercial layer flocks in the Netherlands. Isolation, biochemical identification, species-specific PCR tests and classical serotyping were performed. In addition, molecular typing by Enterobacterial Repetitive Intergenic Consensus-Based Polymerase Chain Reaction (ERIC-PCR) and sequence analysis of the partial HPG2 region of A. paragallinarum were applied and results of both techniques were compared. Moreover, the pathogenicity of an isolate of the most common genotype detected in the Netherlands was determined in an animal experiment. All 18 Avibacterium isolates were nicotinamide adenine dinucleotide-dependent. All isolates were detected by the species-specific conventional PCR while 33% of the isolates were missed by the species-specific real-time PCR. Sequence analysis showed a probe mismatch as a result of a single nucleotide polymorphism (G1516A). Modification of the probe of the real-time PCR was necessary to overcome false negative results. Molecular typing showed that sequence analysis of the partial HPG2 region was in concordance with ERIC-PCR results and indicated the presence of two major genotypes. Serotyping showed the presence of serovars A-1, A-2 and B-1. There was no correlation between genotyping results and serotyping results. Inoculation of an isolate of the most prevalent genotype, and belonging to serovar A-1, into brown layer hens demonstrated the pathogenicity of this isolate.

Surveillance for Avibacterium paragallinarum in autopsy cases of birds from small chicken flocks using a real-time PCR assay.

Infectious coryza is a severe respiratory disease of chickens associated with large economic losses in affected commercial flocks. The fastidious causative pathogen, Avibacterium paragallinarum, is difficult to recover and identify, resulting in delayed diagnosis and enhanced spread of the agent. Small poultry flocks are increasingly common in rural and suburban environments. We assessed the frequency of A. paragallinarum using real-time PCR and clinical conditions present in samples from such flocks submitted to the California Animal Health and Food Safety Laboratory System (Davis, CA) in 2018. From the 294 samples collected for our study, 86 (30%) were PCR-positive for A. paragallinarum. Juvenile birds (≤1 y) were significantly more likely to be PCR-positive ( p = 0.017), and birds diagnosed with respiratory disease had lower Ct values ( p = 0.001) than those without. Concurrent infections were also identified, including with Mycoplasma gallisepticum (18.6%), M. synoviae (18.6%), infectious bronchitis virus (12.8%), and infectious laryngotracheitis virus (7.0%). Only 46.5% of PCR-positive chickens had antemortem respiratory signs, making endemic infections in these flocks highly likely. Our study demonstrates that A. paragallinarum is present in small-flock operations including those without respiratory disease and may present a risk for airborne pathogen transmission to commercial poultry operations.

Serotyping and antimicrobial resistance of Mannheimia haemolytica strains from European cattle with bovine respiratory disease.

The objective of this study was to assess the prevalence of three serotypes, A1, A2, and A6 in 98 M. haemolytica isolates collected from clinical BRD cases in European cattle and assess their antimicrobial resistance profiles. Isolates were characterized by serotyping (plate agglutination and serotype specific PCR) and antimicrobial susceptibility testing. The study identified a predominance of serotypes A1 (59%) and A6 (22%) in European M. haemolytica isolates exhibiting a relatively low level of antimicrobial resistance. A comprehensive understanding of the relative prevalence of different M. haemolytica serotypes in Europe informs a targeted approach for vaccine design against BRD.

Prevalence and comparative analysis of the type IV secretion system in Aggregatibacter actinomycetemcomitan.

BACKGROUD/PURPOSE: Aggregatibacter actinomycetemcomitans has emerged as one of the aetiological agents in periodontal disease. Although Type IV secretion systems (T4SSs) are widely distributed in many bacteria, the genetic features and distribution of T4SSs in A. actinomycetemcomitans remain unclear. In this study, we investigated the prevalence of A. actinomycetemcomitans serotypes and their T4SSs in a Taiwanese population. METHODS: A comparative analysis of 20 A. actinomycetemcomitans genomes and their T4SSs deposited in GenBank was performed. One hundred subjects, including 20 periodontitis and 80 normal subjects, were enrolled and PCR identification of A. actinomycetemcomitans serotypes and T4SS genes were performed. RESULTS: Of 100 subjects, serotypes C (22%) and E (11%) were most common. In addition, T4SSs were distributed in all of the serotypes. The prevalence of T4SSs and their location in plasmids in periodontitis subjects were 1.28-2 fold higher but not significantly different compared to normal subjects. Of 20 A. actinomycetemcomitans genomes, only ten with complete T4SS modules could be detected, which was highly correlated with localized aggressive periodontitis (p < 0.1). Nine of ten T4SS modules were from periodontitis subjects. Phylogenetic analysis of 10 T4SSs in A. actinomycetemcomitans showed that they were clustered into two groups, T4SSAaI and T4SSAaII, with only T4SSAaI appearing in the Taiwanese subjects. CONCLUSION: A. actinomycetemcomitans strains with different serotypes carrying T4SSAaI are widely distributed in a Taiwanese population. This is the first report to show the distribution and detailed comparative genomics of T4SSs in A. actinomycetemcomitans.

Treatment history and antimicrobial susceptibility results for Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni isolates from bovine respiratory disease cases submitted to the Iowa State University Veterinary Diagnostic Laboratory from 2013 to 2015.

Bovine respiratory disease is the most costly disease facing the cattle industry. Increasing resistance to antimicrobial treatment has been presented as a significant contributing factor, often through summarized susceptibility testing data. We assessed the relationship between previous antimicrobial treatment and antimicrobial susceptibility results from isolates of Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni cultured from bovine respiratory cases submitted to the Iowa State University Veterinary Diagnostic Laboratory from 2013 to 2015. Antimicrobial susceptibility data from 1,251 bacterial isolates were included for analysis. More bacterial isolates from cattle that received antimicrobial treatment showed resistance compared to isolates from untreated cattle, and the percentage of resistant isolates increased as the number of antimicrobial treatments increased. Resistance to enrofloxacin, spectinomycin, tilmicosin, and tulathromycin was present in >75% of M. haemolytica isolates from cattle that had received 3 or more antimicrobial treatments; resistance to each of those 4 antimicrobials was present in ≤10% of M. haemolytica isolates from untreated cattle. Similar but less dramatic trends were apparent for isolates of P. multocida and H. somni. The percentage of multi-drug resistant bacterial isolates also increased with the number of treatments. Results of our study suggest that previous antimicrobial treatment may have a profound effect on antimicrobial susceptibility testing. Summarized susceptibility results from diagnostic laboratories should not be used to make generalized statements regarding trends in antimicrobial resistance without providing context regarding antimicrobial treatment history.

Aggregatibacter actinomycetemcomitans serotype prevalence and antibiotic resistance in a UK population with periodontitis.

OBJECTIVES: Aggregatibacter actinomycetemcomitans is a recognised pathogen involved in aggressive periodontitis. Seven serotypes of A. actinomycetemcomitans exist with a range of virulence and distribution dependent on ethnicity and geography. The ability of A. actinomycetemcomitans to invade soft tissue can necessitate the use of systemic antibiotics for treatment, however variations in its antibiotic susceptibility exist dependent on geographical location. METHODS: Serotypes of A. actinomycetemcomitans isolates from a UK cohort of 50 patients with aggressive periodontitis were determined by PCR. Resistance of the isolates to eight antibiotics [penicillin (1U), amoxicillin (2µg), amoxicillin/clavulanic acid (30µg), metronidazole (5µg), clindamycin (2µg), tetracycline (10µg), ciprofloxacin (5µg) and ceftazidime (30µg)] were determined by disk diffusion according to BSAC guidelines. RESULTS: Prevalences of serotypes a, c, b, e and mixed serotypes were 48%, 22%, 2%, 2% and 12%, respectively. The serotype of isolates from seven patients (14%) could not be deduced by PCR. Of the 56 isolates tested, 100% were resistant to penicillin and metronidazole, 87.5% to clindamycin, 83.9% to amoxicillin and 76.8% to ceftazidime. Low rates of resistance to tetracycline (8.9% resistant) and amoxicillin/clavulanic acid (14.3% resistant) were observed, whereas no isolates were resistant to ciprofloxacin. CONCLUSIONS: As in a number of publications the suggested treatment of aggressive periodontitis includes the combined use of amoxicillin with metronidazole, these results highlight the need for culture and antimicrobial susceptibility investigations in patients with aggressive periodontitis prior to systemic use of antibiotics concomitantly to periodontal therapy.

Small ruminant pasteurellosis in Tigray region, Ethiopia: marked serotype diversity may affect vaccine efficacy.

The aim of this study was to investigate the prevalent Bibersteinia, Mannheimia and Pasteurella serotypes, risk factors and degree of serotype co-infections in sheep and goats in the Tigray region of Ethiopia. Serum was collected from 384 sheep and goats from the Tanqua-Abergelle district of Tigray region using cross-sectional random sampling. An indirect haemagglutination test was used for serotyping. Risk factors for infections were evaluated by logistic regression. Potential clustering of multiple serotypes within individual animals due to common risk factors was evaluated by redundancy analysis. Eight serotypes were identified: all studied animals were serologically positive for at least one serotype. Overall, 355 (92·45%) of the animals were infected by four or more serotypes. Of the five risk factors studied, peasant association (PA), animal species, age (serotype A1), and bodyweight (serotype T15) were significantly associated with infection, but sex was not significant. Only PA explained a significant proportion of the variation (adjusted R 2 = 0·16) in the serological responses. After the effect of PA was accounted for, T3 and T4; A7 and Pasteurella multocida A; and A7 and T10 were positively correlated for co-infection, while T4 and T10 were less likely to be found within the same animal. Diverse serotypes were circulating in the Tigray region and could be a challenge in selecting serotypes for vaccine.

Molecular epidemiology of an outbreak of clinical mastitis in sheep caused by Mannheimia haemolytica.

The aetiology and epidemiology of outbreaks of clinical mastitis in sheep under extensive pastoral conditions are incompletely understood. The objective of this study was to conduct a detailed investigation of a clinical mastitis outbreak that affected more than 10% of 230 at-risk ewes on a sheep and grain producing property in south east Australia during drought conditions in 2009. Milk samples were collected aseptically from all affected ewes and plated on sheep blood agar for bacterial identification. M. haemolytica was isolated from 80% of the samples that yielded cultivable microorganisms and thus was the main microorganism responsible for the outbreak. Analysis of the restriction endonuclease cleavage patterns of the isolates using pulsed field gel electrophoresis revealed some evidence of clonality, suggesting the possibility of horizontal transmission, but there was also considerable diversity between the clusters of closely related isolates. Multilocus sequence typing of the M. haemolytica isolates revealed most of the isolates belonged to ST1 with no association between the PFGE and MLST fingerprints of the isolates. Resistance to neomycin, streptomycin and sulphafurazole was detected in some of the isolates, but they were all susceptible to penicillin, ampicillin, ceftiofur, amoxycillin/clavulanic acid, ciprofloxacin, tetracycline, erythromycin and trimethoprim. This is the first published record of a comparison of the strains of M. haemolytica involved in a clinical mastitis outbreak in sheep and demonstrates the importance of this pathogen in sheep production systems, particularly during adverse climatic conditions and increased stocking rate.

Porphyromonas gingivalis Fim-A genotype distribution among Colombians.

INTRODUCTION: Porphyromonas gingivalis is associated with periodontitis and exhibit a wide array of virulence factors, including fimbriae which is encoded by the FimA gene representing six known genotypes. OBJETIVE: To identify FimA genotypes of P. gingivalis in subjects from Cali-Colombia, including the co-infection with Aggregatibacter actinomycetemcomitans, Treponema denticola, and Tannerella forsythia. METHODS: Subgingival samples were collected from 151 people exhibiting diverse periodontal condition. The occurrence of P. gingivalis, FimA genotypes and other bacteria was determined by PCR. RESULTS: P. gingivalis was positive in 85 patients. Genotype FimA II was more prevalent without reach significant differences among study groups (54.3%), FimA IV was also prevalent in gingivitis (13.0%). A high correlation (p= 0.000) was found among P. gingivalis, T. denticola, and T. forsythia co-infection. The FimA II genotype correlated with concomitant detection of T. denticola and T. forsythia. CONCLUSIONS: Porphyromonas gingivalis was high even in the healthy group at the study population. A trend toward a greater frequency of FimA II genotype in patients with moderate and severe periodontitis was determined. The FimA II genotype was also associated with increased pocket depth, greater loss of attachment level, and patients co-infected with T. denticola and T. forsythia.

INTRODUCCIÓN: Porphyromonas gingivalis es una bacteria asociada con la periodontitis. Expresa una amplia gama de factores de virulencia, incluyendo las fimbrias, las cuales están codificadas por el gen FimA que representa seis genotipos conocidos. OBJETIVO: Identificar los genotipos de FimA de P. gingivalis en pacientes de Cali - Colombia, incluyendo la co -infección con Aggregatibacter actinomycetemcomitans, Treponema denticola y Tannerella forsythia . MÉTODOS: Se obtuvieron muestras subgingivales de 151 individuos con diferentes diagnósticos periodontales. La ocurrencia de P. gingivalis, los genotipos de FimA y otras bacterias se determinó por PCR. RESULTADOS: Porphyromonas gingivalis fue positiva en 85 pacientes. El genotipo FimA II fue más prevalente, pero no hubo diferencias significativas entre los grupos de estudio (54.3%) , FimA IV fue el más frecuente en la gingivitis (13.0%). Una alta correlación (p= 0.000 ) se encontró entre P. gingivalis , T. denticola y T. forsythia. El genotipo FimA II estuvo correlacionado con la detección de T. denticola y T. forsythia . CONCLUSIONES: Porphyromonas gingivalis tuvo una alta frecuencia incluso en el grupo de individuos sanos. Se encontró una tendencia hacia una mayor frecuencia de FimA II en pacientes con periodontitis moderada y severa. El genotipo FimA II también se asoció con una mayor profundidad de la bolsa, una mayor pérdida de nivel de inserción, y con los pacientes en los que se detectó co - infección con T. denticola y T. forsythia.

Naturally Occurring ß-Nicotinamide Adenine Dinucleotide-Independent Avibacterium paragallinarum Isolate in Peru.

The ß-nicotinamide adenine dinucleotide (NAD) requirement has been considered to be essential for the isolation of the causal agent of infectious coryza, Avibacterium paragallinarum. Nevertheless, NAD-independent reports from South Africa and Mexico dismissed this paradigm. It is now accepted that both NAD-dependent and NAD-independent agents are able to cause infectious coryza and thus belong to the species A. paragallinarum. Here, we report for the first time in Peru a NAD-independent isolate from broiler chickens with typical signs of infectious coryza that have received a trivalent inactivated vaccine against infectious coryza. The isolate was identified based on its morphology, biochemical and serologic tests, and PCR results. Partial 16S rRNA gene sequence analysis confirmed the isolate as A. paragallinarum. There have been no cases of NAD-independent A. paragallinarum isolates reported in South America. Increasing reports around the world highlight not only the need to reconsider the in vitro nutritional requirements of this species for its correct isolation but also the cross-protection conferred by commercial infectious coryza vaccines against NAD-independent isolates.