Haemophilus Parasuis Research Articles

Basic Characterization of Natural Transformation in a Highly Transformable Haemophilus parasuis Strain SC1401.

Haemophilus parasuis causes Glässer's disease and pneumonia, incurring serious economic losses in the porcine industry. In this study, natural competence was investigated in H. parasuis. We found competence genes in H. parasuis homologous to ones in Haemophilus influenzae and a high consensus battery of Sxy-dependent cyclic AMP (cAMP) receptor protein (CRP-S) regulons using bioinformatics. High rates of natural competence were found from the onset of stationary-phase growth condition to mid-stationary phase (OD600 from 0.29 to 1.735); this rapidly dropped off as cells reached mid-stationary phase (OD600 from 1.735 to 1.625). As a whole, bacteria cultured in liquid media were observed to have lower competence levels than those grown on solid media plates. We also revealed that natural transformation in this species is stable after 200 passages and is largely dependent on DNA concentration. Transformation competition experiments showed that heterogeneous DNA cannot outcompete intraspecific natural transformation, suggesting an endogenous uptake sequence or other molecular markers may be important in differentiating heterogeneous DNA. We performed qRT-PCR targeting multiple putative competence genes in an effort to compare bacteria pre-cultured in TSB++ vs. TSA++ and SC1401 vs. SH0165 to determine expression profiles of the homologs of competence-genes in H. influenzae. Taken together, this study is the first to investigate natural transformation in H. parasuis based on a highly naturally transformable strain SC1401.

Characterisation of a novel plasmid containing a florfenicol resistance gene in Haemophilus parasuis

Thirty clinical isolates of H. parasuis from pig farms in eastern China were screened for antimicrobial susceptibility. A novel plasmid, designated pHPSGC, was extracted from one isolate with evidence of resistance (elevated minimum inhibitory concentration) to florfenicol. DNA sequencing demonstrated that pHPSGC (5297 base pairs) contains three open reading frames (ORFs), corresponding to the genes rep, floR and lysR. The rep gene of pHPSGC shared 99% sequence identity with the rep gene of pHPS1019. In addition, the region containing floR and lysR in pHPSGC shared 99% similarity with the corresponding region of pCCK381. pHPSGC may be derived from a recombination event between pHPS1019 and pCCK381. A florfenicol resistance gene in H. parasuis may have been transferred via recombination between different plasmids.

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English

Baicalin modulates NF-\u03baB and NLRP3 inflammasome signaling in porcine aortic vascular endothelial cells Infected by Haemophilus parasuis Causing Gl\xe4sser\u2019s disease

Haemophilus parasuis (H. parasuis) can cause vascular inflammatory injury, but the molecular basis of this effect remains unclear. In this study,we investigated the effect of the anti-inflammatory, anti-microbial and anti-oxidant agent, baicalin, on the nuclear factor (NF)-κB and NLRP3 inflammasome signaling pathway in pig primary aortic vascular endothelial cells. Activation of the NF-κB and NLRP3 inflammasome signaling pathway was induced in H. parasuis-infected cells. However, baicalin reduced the production of reactive oxygen species, apoptosis, and activation of the NF-κB and NLRP3 inflammasome signaling pathway in infected cells. These results revealed that baicalin can inhibit H. parasuis-induced inflammatory responses in porcine aortic vascular endothelial cells, and may thus offer a novel strategy for controlling and treating H. parasuis infection. Furthermore, the results suggest that piglet primary aortic vascular endothelial cells may provide an experimental model for future studies of H. parasuis infection.

Limited Interactions between Streptococcus Suis and Haemophilus Parasuis in In Vitro Co-Infection Studies.

Streptococcus suis and Haemophilus parasuis are normal inhabitants of the porcine upper respiratory tract but are also among the most frequent causes of disease in weaned piglets worldwide, causing inflammatory diseases such as septicemia, meningitis and pneumonia. Using an in vitro model of infection with tracheal epithelial cells or primary alveolar macrophages (PAMs), it was possible to determine the interaction between S. suis serotype 2 and H. parasuis strains with different level of virulence. Within H. parasuis strains, the low-virulence F9 strain showed higher adhesion levels to respiratory epithelial cells and greater association levels to PAMs than the high-virulence Nagasaki strain. Accordingly, the low-virulence F9 strain induced, in general, higher levels of pro-inflammatory cytokines than the virulent Nagasaki strain from both cell types. In general, S. suis adhesion levels to respiratory epithelial cells were similar to H. parasuis Nagasaki strain. Yet, S. suis strains induced a significantly lower level of pro-inflammatory cytokine expression from epithelial cells and PAMs than those observed with both H. parasuis strains. Finally, this study has shown that, overall and under the conditions used in the present study, S. suis and H. parasuis have limited in vitro interactions between them and use probably different host receptors, regardless to their level of virulence.

The effect of rfaD and rfaF of Haemophilus parasuis on lipooligosaccharide induced inflammation by NF-κB/MAPKs signaling in porcine alveolar macrophages.

In Haemophilus parasuis, the rfa cluster has been identified as a virulence-associated factor that is involved in lipooligosaccharide (LOS) biosynthesis. In this study, we assessed the roles of rfaD and rfaF genes in H. parasuis SC096 on LOS-induced pro-inflammatory factors and the related signaling pathways in porcine alveolar macrophages (PAMs) by real-time PCR and western blotting. The results showed that the LOSs of both rfaD and rfaF mutants (ΔrfaD-LOS and ΔrfaF-LOS) significantly decreased the mRNA expression of pro-inflammatory factors (IL-1α, IL-1β, IL-6, IL-8 and TNF-α) in PAMs compared with H. parasuis SC096 LOS (WT-LOS). Furthermore, in ΔrfaD-LOS- and ΔrfaF-LOS-treated cells, IκBα degradation was significantly inhibited and levels of phospho-p65 and phospho-p38 were significantly reduced in PAMs. These findings suggested that the rfaD and rfaF genes mediated LOS induction of pro-inflammatory cytokines in PAMs by regulating the NF-κB and MAPKs signaling pathways during H. parasuis infection.

Tea polyphenols suppress growth and virulence-related factors of Haemophilus parasuis.

The bacterium Haemophilus parasuis (H. parasuis) is the primary cause of Glässer’s disease. Currently, there are no effective vaccines that can confer protection against all H. parasuis serovars. Therefore, the present study aimed to investigate the effect of tea polyphenols on growth, expression of virulence-related factors, and biofilm formation of H. parasuis, as well as to evaluate their protective effects against H. parasuis challenge. Our findings demonstrated that tea polyphenols can inhibit H. parasuis growth in a dose-dependent manner and attenuate the biofilm formation of H. parasuis. In addition, tea polyphenols exerted inhibitory effects on the expression of H. parasuis virulence-related factors. Moreover, tea polyphenols could confer protection against a lethal dose of H. parasuis and can reduce pathological tissue damage induced by H. parasuis. In summary, our findings demonstrated the promising use of tea polyphenols as a novel treatment for H. parasuis infection in pigs.

OxyR of Haemophilus parasuis is a global transcriptional regulator important in oxidative stress resistance and growth

Haemophilus parasuis is an opportunistic pathogen and the causative agent of Glässer's disease in swine. This disease has high morbidity and mortality rates in swine populations, and is responsible for major economic losses worldwide. Survival of H. parasuis within the host requires mechanisms for coping with oxidative stress conditions. In many bacteria, OxyR is known to mediate protection against oxidative stress; however, little is known about the role of OxyR in H. parasuis. In the current study, an oxyR mutant strain was constructed in H. parasuis strain SC1401 and designated H. parasuis SC1401∆oxyR. The oxyR mutant strain had a slower growth rate and impaired biofilm formation compared to the wild type strain. Complementation restored the growth-associated phenotypes to wild type levels. Oxidative stress susceptibility testing, using a range of concentrations of H2O2, indicated that H. parasuis SC1401∆oxyR was more sensitive to oxidative stress than the wild type strain. RNA sequencing transcriptome analysis comparing H. parasuis SC1401 with H. parasuis SC1401∆oxyR identified 466 differentially expressed genes. These genes were involved in a wide range of biological processes, including: oxidative stress, transcriptional regulation, and DNA replication, recombination, and repair. These findings provide a foundation for future research to examine the role of OxyR as a global transcriptional regulator and to better define its role in oxidative stress resistance in H. parasuis.

The pharmacokinetic-pharmacodynamic modeling and cut-off values of tildipirosin against Haemophilus parasuis.

The goal of this study was to establish the epidemiological, pharmacodynamic cut-off values, optimal dose regimens for tildipirosin against Haemophilus parasuis. The minimum inhibitory concentrations (MIC) of 164 HPS isolates were determined and SH0165 whose MIC (2 μg/ml ) were selected for PD analysis. The ex vivo MIC in plasma of SH0165 was 0.25 μg/ml which was 8 times lower than that in TSB. The bacteriostatic, bactericidal and elimination activity (AUC24h/MIC) in serum were 26.35, 52.27 and 73.29 h based on the inhibitory sigmoid Emax modeling. The present study demonstrates that 97.9% of the wild-type (WT) isolates were covered when the epidemiological cut-off value (ECV) was set at 8 μg/ml. The parameters including AUC24h, AUC, T1/2, Cmax, CLb and MRT in PELF were 19.56, 60.41, 2.32, 4.02, 56.6, and 2.63 times than those in plasma, respectively. Regarding the Monte Carlo simulation, the COPD was defined as 0.5 μg/ml in vitro, and the optimal doses to achieve bacteriostatic, bactericidal and elimination effect were 1.85, 3.67 and 5.16 mg/kg for 50% target, respectively, and 2.07, 4.17 and 5.78 mg/kg for 90% target, respectively. The results of this study offer a more optimised alternative for clinical use and demonstrated that 4.17 mg/kg of tildipirosin by intramuscular injection could have an effect on bactericidal activity against HPS. These values are of great significance for the effective treatment of HPS infections, but it also be deserved to be validated in clinical practice in the future research.

Coinfection modulates inflammatory responses, clinical outcome and pathogen load of H1N1 swine influenza virus and Haemophilus parasuis infections in pigs

BackgroundRespiratory co-infections are important factor affecting the profitability of pigs production. Swine influenza virus (SIV) may predispose to secondary infection. Haemophilus parasuis (Hps) can be a primary pathogen or be associated with other pathogens such as SIV. To date, little is known about the effect of coinfection with SIV and Hps on the disease severity and inflammatory response and the role of Hps in the induction of pneumonia in the absence of other respiratory pathogens. In the study we investigated the influence of SIV and Hps coinfection on clinical course, inflammatory response, pathogens shedding and load at various time points following intranasal inoculation. The correlation between local concentration of cytokines and severity of disease as well as serum acute phase proteins (APP) concentration has been also studied.ResultsAll co-infected pigs had fever, while in single inoculated pigs fever was observed only in part of animals. Necropsy revealed lesions in the lungs all SIV-inoculated and co-inoculated pigs, while in Hps-single inoculated animals only 1 out of 11 pigs revealed gross lung lesions. The SIV shedding was the highest in co-inoculated pigs. There were no differences between Hps-single inoculated and co-inoculated groups with regard to Hps shedding. The significant increase in Hps titre in the lung has been found only in co-inoculated group. All APP increased after co-infection. In single-inoculated animals various kinetics of APP response has been observed. The lung concentrations of cytokines were induced mostly in SIV + Hps pigs in the apical and middle lobe. These results correlated well with localization of gross lung lesions.ConclusionsThe results revealed that SIV increased the severity of lung lesions and facilitated Hps (PIWetHps192/2015) replication in the porcine lung. Furthermore, Hps influenced the SIV nasal shedding. Enhanced Hps and SIV replication, together with stronger systemic and local inflammatory response contributed to a more severe clinical signs and stronger, earlier immune response in co-inoculated animals. We confirmed the previous evidence that single-Hps infection does not produce significant pneumonic lesions but it should be in mind that other strains of Hps may produce lesions different from that reported in the present study.

Coinfection with Haemophilus parasuis serovar 4 increases the virulence of porcine circovirus type 2 in piglets

BackgroundPostweaning multisystemic wasting syndrome (PMWS) is an emerging disease in swine. Pigs with PMWS are often infected with a variety of other pathogens, including bacteria, viruses and mycoplasm, in addition to porcine circovirus type 2 (PCV2). PCV2 and Haemophilus parasuis serovar 4 (HPS4) coinfection remain epidemic in China.MethodsHere we report construction of a three-week-old naturally farrowed, colostrum-deprived (NFCD) piglet’s infection model and demonstrate that PCV2-infected piglets with the HPS4 coinfection increased the virulence of PCV2 and these pathogens interact acquired PMWS.ResultsAll the single infected piglets were transiently bacteremic or viremic. All the PCV2/HPS4 coinfected piglets developed PMWS, characterized by dyspnea, anorexia, prostration and lose weight severely. Co-infection with PCV2 and HPS4 resulted in an increased amount of virus in serum and tissues, presented a slower generation and lower levels of antibodies against PCV2. Co-infection with PCV2 and HPS4 resulted in further reductions in total and differential peripheral blood leukocyte counts. Meantime, PCV2/ HPS4 coinfection potentiated the severity of lung and lymphoid lesions by PCV2-associated, increased the virulence of PCV2-antigen and enhanced the incidence of PMWS in piglets.ConclusionCo-infection with PCV2 and HPS4 induce the exacerbation of system injuries and enhance the pathogenicity of PCV2 in piglets.

Tilmicosin- and florfenicol-loaded hydrogenated castor oil-solid lipid nanoparticles to pigs: Combined antibacterial activities and pharmacokinetics.

The combined antibacterial effects of tilmicosin (TIL) and florfenicol (FF) against Actinobacillus pleuropneumoniae (APP) (n=2), Streptococcus suis (S.suis) (n=2), and Haemophilus parasuis (HPS) (n=2) were evaluated by chekerboard test and time-kill assays. The pharmacokinetics (PKs) of TIL- and FF-loaded hydrogenated castor oil (HCO)-solid lipid nanoparticles (SLN) were performed in healthy pigs. The results indicated that TIL and FF showed synergistic or additive antibacterial activities against APP, S.suis and HPS with the fractional inhibitory concentration (FIC) ranging from 0.375 to 0.75. The time-kill assays showed that 1/2 minimum inhibitory concentration (MIC) TIL combined with 1/2 MIC FF had a stronger ability to inhibit the growth of APP, S.suis, and HPS than 1 MIC TIL or 1 MIC FF, respectively. After oral administration, plasma TIL and FF concentrations could maintain about 0.1μg/ml for 192 and 176hr. The SLN prolonged the last time point with detectable concentrations (Tlast ), area under the concentration-time curve (AUC0-t ), elimination half-life (T½ke ), and mean residence time (MRT) by 3.1, 5.6, 12.7, 3.4-fold of the active pharmaceutical ingredient (API) of TIL and 11.8, 16.5, 18.1, 12.1-fold of the API of FF, respectively. This study suggests that the TIL-FF-SLN could be a useful oral formulation for the treatment of APP, S.suis, and HPS infection in pigs.

Rapid detection of Haemophilus parasuis using cross-priming amplification and vertical flow visualization

Haemophilus parasuis infection is of considerable economic importance in the swine industry due to high morbidity and mortality in naive swine populations. Accurate detection and identification of the causative agent are difficult, yet necessary, for disease control. In this study, a simple and rapid method of cross-priming amplification (CPA) with a vertical flow (VF) visualization strip was established to detect H. parasuis. The reaction can specifically identify 15 serovar reference strains and 57 clinically isolated strains of H. parasuis, with a detection limit of 14CFU. The performance of the CPA-VF assay was evaluated and compared with that of species-specific PCR by testing 62 clinical culture-positive specimens of H. parasuis. The entire process, from specimen processing to analysis of the results, can be completed in 2h without a complicated apparatus. The convenience and speed of the CPA-VF assay in this study make it a suitable choice for epidemiological investigation and point-of-care testing (POCT) for H. parasuis infection.

Antimicrobial susceptibility patterns of Brazilian Haemophilus parasuis field isolates

ABSTRACT: Haemophilus parasuis is the etiological agent of Glässer’s disease (GD), an ubiquitous infection of swine characterized by systemic fibrinous polyserositis, polyarthritis and meningitis. Intensive use of antimicrobial agents in swine husbandries during the last years triggered the development of antibiotic resistances in bacterial pathogens. Thus, regular susceptibility testing is crucial to ensure efficacy of different antimicrobial agents to this porcine pathogen. In this study, 50 clinical isolates from South Brazilian pig herds were characterized and analyzed for their susceptibility to commonly used antibiotic. The identification and typing of clinical isolates was carried out by a modified indirect hemagglutination assay combined with a multiplex PCR. The susceptibility of each isolate was analyzed by broth microdilution method against a panel of 21 antimicrobial compounds. We found that field isolates are highly resistance to gentamycin, bacitracin, lincomycin and tiamulin, but sensitive to ampicillin, clindamycin, neomycin, penicillin, danofloxacin and enrofloxacin. Furthermore, an individual susceptibility analysis indicated that enrofloxacin is effective to treat clinical isolates with the exception of those classified as serovar 1. The results presented here firstly demonstrate the susceptibility of Brazilian clinical isolates of H. parasuis to antimicrobials widely used by swine veterinary practitioners and strengthen the need to perform susceptibility test prior to antibiotic therapy during GD outbreaks. In addition, because only six antimicrobial drugs (28.6%) were found effective against field isolates, a continuous surveillance of the susceptibility profile should be of major concern to the swine industry.

Haemophilus parasuis infection activates NOD1/2-RIP2 signaling pathway in PK-15 cells

Haemophilus parasuis, an important swine pathogen, was recently proven able to invade into endothelial or epithelial cell in vitro. NOD1/2 are specialized NLRs that participate in the recognition of pathogens able to invade intracellularly and therefore, we assessed that the contribution of NOD1/2 to inflammation responses during H. parasuis infection. We observed that H. parasuis infection enhanced NOD2 expression and RIP2 phosphorylation in porcine kidney 15 cells. Our results also showed that knock down of NOD1/2 or RIP2 expression respectively significantly decreased H. parasuis-induced NF-κB activity, while the phosphorylation level of p38, JNK or ERK was not changed. Moreover, real-time PCR result showed that NOD1, NOD2 or RIP2 was involved in the expression of CCL4, CCL5 and IL-8. Inhibition of NOD1 and NOD2 significantly reduced CCL5 promoter activity, even in a more effective way compared with inhibition of TLR.

Haemophilus parasuis CpxRA two-component system confers bacterial tolerance to environmental stresses and macrolide resistance

Haemophilus parasuis is an opportunistic pathogen localized in the upper respiratory tracts of pigs, its infection begins from bacterial survival under complex conditions, like hyperosmosis, oxidative stress, phagocytosis, and sometimes antibiotics as well. The two-component signal transduction (TCST) system serves as a common stimulus-response mechanism that allows microbes to sense and respond to diverse environmental conditions via a series of phosphorylation reactions. In this study, we investigated the role of TCST system CpxRA in H. parasuis in response to different environmental stimuli by constructing the ΔcpxA and ΔcpxR single deletion mutants as well as the ΔcpxRA double deletion mutant from H. parasuis serotype 4 isolate JS0135. We demonstrated that H. parasuis TCST system CpxRA confers bacterial tolerance to stresses and bactericidal antibiotics. The CpxR was found to play essential roles in mediating oxidative stress, osmotic stresses and alkaline pH stress tolerance, as well as macrolide resistance (i.e. erythromycin), but the CpxA deletion did not decrease bacterial resistance to abovementioned stresses. Moreover, we found via RT-qPCR approach that HAPS_RS00160 and HAPS_RS09425, both encoding multidrug efflux pumps, were significantly decreased in erythromycin challenged ΔcpxR and ΔcpxRA mutants compared with wild-type strain JS0135. These findings characterize the role of the TCST system CpxRA in H. parasuis conferring stress response tolerance and bactericidal resistance, which will deepen our understanding of the pathogenic mechanism in H. parasuis.

Development of Serotype-Specific PCR Assays for Typing of Haemophilus parasuis Isolates Circulating in Southern China.

The bacterium Haemophilus parasuis is the specific pathogenic cause of Glässer's disease in swine. Fifteen serotypes of H. parasuis have been reported. A method to serotype H. parasuis isolates accurately would help to prevent and control Glässer's disease outbreaks through appropriate vaccination and to understand the epidemiology in specific geographic areas. However, according to traditional serotyping, the rate of nontypeable (NT) strains is 10 to 40%, which gives low accuracy. In the present study, we developed a set of PCR assays that are able to identify all the currently known H. parasuis serotypes, with a detection limit of 5 CFU. This PCR method is particularly useful to distinguish serotype 5 from serotype 12. We then surveyed the serotype prevalence of H. parasuis isolates from southern China using both the traditional indirect hemagglutination (IHA) and current PCR methods. Of the 298 isolates tested, 228 (76.51%) and 281 (94.30%) were serotyped by the IHA and PCR tests, respectively, with a concordance rate of 80.87% (241/298). The most prevalent serotypes obtained by PCR were 4, 5, 12, 13, NT, and 2, and the most prevalent obtained by IHA were NT, 5, 4, 12, 13, and 2. In conclusion, the PCR assays developed in this study provide a rapid and specific method for the molecular serotyping of H. parasuis.

New insights about functional and cross-reactive properties of antibodies generated against recombinant TbpBs of Haemophilus parasuis

Vaccines have become fundamental in the control and elimination of Glässer Disease, a systemic disease of pigs caused by Haemophilus parasuis. The classic vaccines available for prevention of this infection were developed without a robust knowledge about host immunological mechanisms. In this study, we demonstrated the presence of cross-reactive epitopes on both the N-lobe and C-lobe of variants of transferrin binding protein B (TbpBs) expressed on the surface of 6 virulent serovars of H. parasuis. Antibodies against TbpB-derived antigens were capable of increasing the phagocytic capacity of neutrophils and were also capable of blocking porcine transferrin from binding to TbpB. Surprisingly, none of the pig or mice antisera from animals immunized with TbpB-derived antigens mixed with Montanide IMS 2215 VG PR adjuvant were able to activate the classical complement pathway (CCP). In contrast, antisera from mice immunized with TbpB-derived antigens adjuvanted with Freund’s adjuvants or Montanide Gel 01 were able to activate the CCP and kill H. parasuis. Our results demonstrate that the type of adjuvant can modulate the functional response induced by TbpB-derived antigens. Based on these results, we propose that a properly formulated TbpB-based vaccine may elicit a functional protective antibody response with broad cross-reactivity against heterologous strains of H. parasuis.

Comparative transcriptional profiling of tildipirosin-resistant and sensitive Haemophilus parasuis

Numerous studies have been conducted to examine the molecular mechanism of Haemophilus parasuis resistance to antibiotic, but rarely to tildipirosin. In the current study, transcriptional profiling was applied to analyse the variation in gene expression of JS0135 and tildipirosin-resistant JS32. The growth curves showed that JS32 had a higher growth rate but fewer bacteria than JS0135. The cell membranes of JS32 and a resistant clinical isolate (HB32) were observed to be smoother than those of JS0135. From the comparative gene expression profile 349 up- and 113 downregulated genes were observed, covering 37 GO and 63 KEGG pathways which are involved in biological processes (11), cellular components (17), molecular function (9), cellular processes (1), environmental information processing (4), genetic information processing (9) and metabolism (49) affected in JS32. In addition, the relative overexpression of genes of the metabolism pathway (HAPS_RS09315, HAPS_RS09320), ribosomes (HAPS_RS07815) and ABC transporters (HAPS_RS10945) was detected, particularly the metabolism pathway, and verified with RT-qPCR. Collectively, the gene expression profile in connection with tildipirosin resistance factors revealed unique and highly resistant determinants of H. parasuis to macrolides that warrant further attention due to the significant threat of bacterial resistance.

Molecular study of an outer fragment of Haemophilus parasuis neuraminidase and utility with diagnostic and immunogen purposes

Haemophilus parasuis is a swine pathogenic organism, being the causative agent of Glässer's disease. It has got some virulence factors, some of which act as potential candidates for the vaccine developing. Among them there is the neuraminidase enzyme, which is located inside the outer membrane and contains a β-barrel domain with seven external loops. By using the polymerase chain reaction technique, the β-barrel fragment was amplified, sequenced and analysed for the 15 H. parasuis reference serotypes. The results showed a small diversity for them, except for serotype 2, which has a deletion that covers the loops with potential to be used as vaccine antigen. However, some of the other serotypes showed the same nucleotidic sequence between them, such those 6 and 7 or those 12 and 13. This fact was also confirmed by means of phylogenetic analysis. For these reasons, the tested fragment might result in a putative candidate for the development of subunit vaccines against all the serotypes causing Glässer's disease outbreaks, with the exception of serotype 2, alone or in combination with other proven immunogenicity molecules. Anyway, further studies should be carried out in pigs in order to confirm this hypothesis. Finally, this outer fragment of H. parasuis neuraminidase could be used as a suitable diagnostic tool at a species level, for instance, by PCR.