Human granulocytic anaplasmosis with rash and rhabdomyolysis: A case report.

Human granulocytic anaplasmosis (HGA) is a tick-borne infection caused by Anaplasma phagocytophilum. Only seven cases of HGA have been reported in Japan to date. We report the case of a 61-year-old female farmer who developed HGA with rash and rhabdomyolysis. The patient had fever and erythema covering the entire body, including the palms. An induration with an eschar was observed on the right leg, indicating that the patient had been bitten by a tick. Elevated serum creatinine and creatinine kinase levels and hematuria indicated rhabdomyolysis. We suspected Japanese spotted fever, a tick-borne illness caused by Rickettsia Japonica, and administered minocycline and ciprofloxacin for a week. Transient neutropenia and thrombocytopenia were observed, but the symptoms improved. Polymerase chain reaction (PCR) and antibody tests for R. japonica and Orientia tsutsugamushi, which causes scrub typhus, were both negative. The PCR test for severe fever with thrombocytopenia syndrome virus was also negative. Antibodies against A. phagocytophilum-related proteins were detected by western blotting, indicating seroconversion of IgG with paired serum samples, and the patient was diagnosed with HGA. HGA should be suspected in acute febrile patients with a history of outdoor activity and cytopenia, with or without a rash. A testing system and the accumulation of cases in Japan are necessary for the early diagnosis and appropriate treatment of HGA.

Co-infection of tick-borne bacterial pathogens in ticks in Inner Mongolia, China.

Tick-borne infectious diseases pose a serious health threat in certain regions of the world. Emerging infectious diseases caused by novel tick-borne pathogens have been reported that are causing particular concern. Several tick-borne diseases often coexist in the same foci, and a single vector tick can transmit two or more pathogens at the same time, which greatly increases the probability of co-infection in host animals and humans and can lead to an epidemic of tick-borne disease. The lack of epidemiological data and information on the specific clinical symptoms related to co-infection with tick-borne pathogens means that it is not currently possible to accurately and rapidly distinguish between a single pathogen infection and co-infection with multiple pathogens, which can have serious consequences. Inner Mongolia in the north of China is endemic for tick-borne infectious diseases, especially in the eastern forest region. Previous studies have found that more than 10% of co-infections were in host-seeking ticks. However, the lack of data on the specific types of co-infection with pathogens makes clinical treatment difficult. In our study, we present data on the co-infection types and the differences in co-infection among different ecological regions through genetic analysis of tick samples collected throughout Inner Mongolia. Our findings may aid clinicians in the diagnosis of concomitant tick-borne infectious diseases.

Serologic Evidence of Human Exposure to Ehrlichiosis Agents in Japan.

In retrospective analyses, we report 3 febrile patients in Japan who had seroconversion to antibodies against Ehrlichia chaffeensis antigens detected by using an immunofluorescence and Western blot. Our results provide evidence of autochthonous human ehrlichiosis cases and indicate ehrlichiosis should be considered a potential cause of febrile illness in Japan.

Surveillance of Borrelia miyamotoi-carrying ticks and genomic analysis of isolates in Inner Mongolia, China.

BACKGROUND: Borrelia miyamotoi is a newly described relapsing fever spirochete transmitted by ixodid tick species. Little is known about the prevalence of B. miyamotoi infections in humans and ticks in Inner Mongolia, China. Therefore, we investigated the prevalence of B. miyamotoi in Ixodes persulcatus ticks, and we aimed to isolateB. miyamotoi from I. persulcatus from four regions of Greater Khingan, Inner Mongolia, China. METHODS: From May to June each year during the period 2016-2019, host-seeking adult I. persulcatus ticks were collected from vegetation. Genomic DNA was prepared from half of each tick body for PCR template, and the remaining half was used to cultivate B. miyamotoi in BSK-M medium. We employed quantitative real-time PCR (qPCR) to detect Borrelia DNA in the ticks and to calculate the prevalence of B. miyamotoi and infections with other borreliae. For characterization of the isolated B. miyamotoi, we performed draft genome sequencing and multilocus sequencing analysis (MLSA). RESULTS: A total of 2656 adult I. persulcatus ticks were collected. The overall prevalence of relapsing fever (RF) borreliae in ticks was 5.0% (134/2656) and that of Lyme disease (LD) borreliae was 43.8% (1164/2656). Co-infection with RF and LD borreliae was observed in 63 ticks (2.4%). Ticks that were positive for RF borreliae by qPCR were subjected to glycerophosphodiester diester phosphodiesterase gene (glpQ) PCR amplification and sequencing, through which we identified the RF borrelia specimens as B. miyamotoi. Furthermore, the B. miyamotoi strain Hetao-1 was isolated from I. persulcatus, and a draft genome sequence was obtained from the isolate. Sequencing determined the strain Hetao-1 genome to be approximately 906.1 kbp in length (28.9% average GC content), and MLSA identified the strain as ST633, which has previously been reported in Japan and Mongolia. CONCLUSION: We detected B. miyamotoi from I. persulcatus ticks collected in Inner Mongolia, and successfully isolated a B. miyamotoi strain. To our knowledge, this is the first study to culture a B. miyamotoi isolate from China. The data on the prevalence of B. miyamotoi and other borreliae in I. persulcatus ticks will be fundamental for future epidemiological studies of B. miyamotoi disease in Inner Mongolia.

Diversity unearthed by the estimated molecular phylogeny and ecologically quantitative characteristics of uncultured Ehrlichia bacteria in Haemaphysalis ticks, Japan.

Ehrlichia species are obligatory intracellular bacteria transmitted by arthropods, and some of these species cause febrile diseases in humans and livestock. Genome sequencing has only been performed with cultured Ehrlichia species, and the taxonomic status of such ehrlichiae has been estimated by core genome-based phylogenetic analysis. However, many uncultured ehrlichiae exist in nature throughout the world, including Japan. This study aimed to conduct a molecular-based taxonomic and ecological characterization of uncultured Ehrlichia species or genotypes from ticks in Japan. We first surveyed 616 Haemaphysalis ticks by p28-PCR screening and analyzed five additional housekeeping genes (16S rRNA, groEL, gltA, ftsZ, and rpoB) from 11 p28-PCR-positive ticks. Phylogenetic analyses of the respective genes showed similar trees but with some differences. Furthermore, we found that V1 in the V1-V9 regions of Ehrlichia 16S rRNA exhibited the greatest variability. From an ecological viewpoint, the amounts of ehrlichiae in a single tick were found to equal approx. 6.3E+3 to 2.0E+6. Subsequently, core-partial-RGGFR-based phylogenetic analysis based on the concatenated sequences of the five housekeeping loci revealed six Ehrlichia genotypes, which included potentially new Ehrlichia species. Thus, our approach contributes to the taxonomic profiling and ecological quantitative analysis of uncultured or unidentified Ehrlichia species or genotypes worldwide.

Growth Characteristics of Rickettsia Species LON Strains Closely Related to Rickettsia japonica Isolated from Haemaphysalis longicornis Ticks in Mouse Derived L929 and Human-Derived THP-1 Host Cell Lines.

Non-pathogenic Rickettsia species LON strains closely related to an agent of Japanese spotted fever (JSF), R. japonica, were isolated in Japan from Haemaphysalis longicornis ticks in 2001. However, the biological properties of LONs in mammalian host cells are poorly understood. In this study, microscopic analysis showed that LONs in a mouse-derived L929 host cell line were rod shaped with sizes of 0.3-0.5 × 0.5-2.0 µm. Molecular analysis revealed the existence of a LON-specific disrupted open reading frame in R. japonica-related group-specific DNA regions. Growth kinetics of LON-2 and LON-13 strains analyzed by a quantitative real-time PCR showed 100-fold or more increment of LONs cultured in L929 host cells at 30°C and slightly less increment at 33°C, and 25-fold increment in human-derived THP-1 host cells at 35°C on day 7 (168 h) post infection. The generation times of the two LON strains cultured in L929 and THP-1 were estimated to be 9.4-12.9 h and 9.6-10.9 h, respectively. To our knowledge, this is the first report on the biological characteristics of Rickettsia sp. LON strains in mammalian cells, which may provide significant information for the experimental approaches for other rickettsiae.

Influence of Muscle Fiber Direction on Migration of Salmonella Enteritidis, Staphylococcus aureus, and Escherichia coli into Raw Chicken Breast.

ABSTRACT: The influence of muscle fiber direction (parallel or perpendicular) in relation to the inoculation surface on migration of Salmonella Enteritidis, Staphylococcus aureus, and Escherichia coli into raw chicken breasts was examined. Chicken breast samples with two types of surface fibers (running parallel or perpendicular to the surface) were inoculated with cultures of each bacterium. Inoculated samples were stored for 5 min, 1 h, or 24 h at 4°C. After storage, the samples were divided into segments, and bacterial counts were determined in different regions (inoculation surface, inoculation surface to 1 cm, 1 to 2 cm, 2 to 4 cm, and 4 to 6 cm). The migration of bacteria did not change at 5 min or 1 h regardless of fiber direction. However, after 24 h each bacterium was detected at 4 to 6 cm in the pieces of sample with a perpendicular muscle fiber surface cut. Although these bacteria were detected at 4 to 6 cm in samples with muscle fibers perpendicular to the inoculated surface, these results do not clearly indicate that bacteria migrated into the chicken breast. To monitor actual migration of bacteria into the chicken breast, the tops of the perpendicular muscle fibers of the breast sample were inoculated with bioluminescent E. coli Xen-14. Various regions of the breast sample (inoculation surface and cut surfaces at 1, 2, 4, and 6 cm) were stamped directly on growth medium. Culture revealed that the bacteria migrated directly under the contaminated site and dispersed along the surface of the chicken breast segments. More bacteria distributed laterally than migrated directly below the contamination site. These results suggest that the direction of the muscle fibers is a major factor influencing migration of pathogenic bacteria into chicken breast.

Effect of (-)-Epigallocatechin Gallate to Staphylococcal Enterotoxin A on Toxin Activity.

Staphylococcal enterotoxin A (SEA) functions both as superantigens that stimulate non-specific T cell proliferation as well as potent gastrointestinal toxins. We previously reported that (-)-epigallocatechin gallate (EGCG) binds to SEA. Therefore, the ability of EGCG to inhibit SEA toxin activity was examined. As a result, EGCG significantly decreased SEA-induced expression and production of interferon gamma (IFN-γ). In addition, EGCG inhibited SEA-induced spleen cell proliferation. To investigate the role of the galloyl group in EGCG on SEA cytotoxicity in more detail, the effect of the binding of a hydroxyl group at position 3 of the galloyl group in EGCG to SEA on SEA cytotoxicity was examined using two methylated EGCG. SEA cytotoxicity was significantly controlled in both (-)-3''-Me-EGCG and (-)-4''-Me-EGCG. These results suggest that EGCG inhibits toxic activity via direct interaction with SEA or without any interaction with SEA. The binding affinity between SEA and EGCG under in vivo conditions was examined using a model solution. Although after treatment under acidic and alkaline conditions, the presence of protein and the digestive tract model solution, EGCG still interacted with SEA. Our studies are the first to demonstrate the effect of the binding of EGCG to SEA on toxin activity.

Intracellular proliferation of Anaplasma phagocytophilum is promoted via modulation of endoplasmic reticulum stress signaling in host cells.

Anaplasma phagocytophilum, an obligate intracellular bacterium that propagates within host granulocytes, is considered to modify the host intracellular environment for pathogenesis. However, the mechanism(s) underlying such host modifications remain unclear. Here, we aimed to investigate the relation between A. phagocytophilum and endoplasmic reticulum (ER) stress in THP-1 cells. A. phagocytophilum activated the three ER stress sensors: inositol-requiring enzyme-1 (IRE1), protein kinase RNA-like endoplasmic reticulum kinase (PERK), and activating transcription factor-6 (ATF6). IRE1 activation occurred immediately after host cell invasion by A. phagocytophilum; however, the activated IRE1-induced splicing of X-box-binding protein 1 was not promoted during A. phagocytophilum infection. This suppression was sustained even after the doxycycline-mediated elimination of intracellular A. phagocytophilum. IRE1 knockdown accelerated A. phagocytophilum-induced apoptosis and decreased intracellular A. phagocytophilum. These data suggest that A. phagocytophilum utilizes IRE1 activation to promote its own intracellular proliferation. Moreover, PERK and ATF6 partially mediated A. phagocytophilum-induced apoptosis by promoting the expression of CCAAT/enhancer-binding protein homologous protein, which induces the transcription of several proapoptotic genes. Thus, A. phagocytophilum possibly manipulates the host ER stress signals to facilitate intracellular proliferation and infection of surrounding cells before/after host cell apoptosis.

Insight of diagnostic performance using B-cell epitope antigens derived from triple P44-related proteins of Anaplasma phagocytophilum.

Human granulocytic anaplasmosis (HGA) is caused by Anaplasma phagocytophilum. Indirect immunofluorescence assay (IFA) is generally used for HGA serodiagnosis. A. phagocytophilum immunodominant P44 major outer membrane proteins are encoded by p44/msp2 multigene family, responsible for IFA reactivity. However, because multiple P44-related proteins may involve immunoreactivity in IFA, the available diagnostic antigens remain obscure. In this study, we identified 12 B-cell epitopes on triple P44-related proteins using peptide array that reacted with 4 HGA patients' sera. Then, peptide spot immunoassay using 14 synthetic peptides derived from those 12 epitopes as antigens was applied for the detection of antibody to A. phagocytophilum from patients with fever of unknown origin. The sensitivities and diagnostic efficiencies of this immunoassay were higher than those of Western blot analysis using 3 recombinant proteins previously developed. Thus, the immunoassay using our epitope-derived antigens, which has higher diagnostic performances, may have significant benefit for HGA serodiagnosis.

Molecular Detection and Characterization of p44/msp2 Multigene Family of Anaplasma phagocytophilum from Haemaphysalis longicornis in Mie Prefecture, Japan.

Anaplasma phagocytophilum is an obligate intracellular bacterium that causes human granulocytic anaplasmosis (HGA), an emerging tick-borne infectious disease. This bacterium expresses various 44-kDa major outer membrane proteins encoded by the p44/msp2 multigene family to avoid the host immune system. We previously detected A. phagocytophilum p44/msp2 from the tick Haemaphysalis longicornis in Mie Prefecture, Japan in 2008. In this study, we further investigated a total of 483 H. longicornis ticks (220 adults and 263 nymphs) collected from the Mie Prefecture by PCR targeting p44/msp2 to characterize the p44/msp2 multigene family of A. phagocytophilum. Six of the 483 ticks tested were PCR-positive for A. phagocytophilum p44/msp2, and these positive individuals were at the nymph stage of the tick life cycle. Cloning, sequencing, and phylogenetic analyses of the amplicons revealed that the 11 p44/msp2 clones obtained from the positive ticks shared a 54.9%-99.3% amino acid sequence similarity with the 27 previously identified clones from HGA patients in Japan. In particular, 6 p44/msp2 clones displayed the highest similarities (97.2%-99.3%) with 3 previously identified clones (FJ417343, FJ417345, FJ417357). Thus, the data from this study provide important public health information regarding A. phagocytophilum infection transmitted by H. longicornis ticks, especially at the nymph stage.

Predominant Shift of Different P44-Expressing Anaplasma phagocytophilum in Infected HL-60, THP-1, NB4, and RF/6A Cell Lines.

Anaplasma phagocytophilum, an agent of human granulocytic anaplasmosis, is an obligatory intracellular bacterium that dominantly produces P44 outer membrane proteins encoded by the p44/msp2 multigene family, which are major antigens for serodiagnosis. However, A. phagocytophilum antigens from cultures with different cell lines seem to have varying reactivities with sera. In this study, we performed RNA-seq to investigate the P44 expression of A. phagocytophilum propagated in 4 cell lines. In infected HL-60 cells, the P44-2b transcript was predominant in the first RNA-seq analysis (HL-60.1). However, the P44-23 transcript was predominant in the second RNA-seq analysis at 1 month after additional passages (HL-60.2). We further analyzed the P44 expression of A. phagocytophilum cultured in THP-1, NB4, and RF/6A cells through consecutive passages in the same cell lines for 1 year after transferring A. phagocytophilum from infected HL-60 cells to the respective cell lines. In the long-term cultures, P44-18, P44-78, and P44-51 were predominantly transcribed in infected THP-1, NB4, and RF/6A cells, respectively. Therefore, the predominant shifts of different P44-expressing transcripts of A. phagocytophilum might occur during cell culture even in the same cell line at different time points of sample harvest (HL-60.1 and HL-60.2), which may be attributed to host cell adaptation/selection/interaction.

Isolation and molecular detection of Ehrlichia species from ticks in western, central, and eastern Japan.

Ehrlichiosis is a tick-borne bacterial disease caused by pathogens of the Ehrlichia genus. Although human ehrlichiosis has not been reported in Japan, Ehrlichia spp., which are closely related to Ehrlichia chaffeensis, were detected in several species of ixodid ticks. In this study, the presence of Ehrlichia spp. in ticks in Japan was studied by using isolation and molecular detection methods. In total, 1237 ticks were collected from vegetation in western, central, and eastern parts of Japan. The ticks were tested for detection of ehrlichial DNA with a nested polymerase chain reaction and/or isolation by inoculation of mice with the homogenate. Ehrlichial DNA was detected in 29 of these ticks. The ehrlichial DNAs, groEL and 16S rRNA genes, detected in Ixodes turdus showed a high similarity to those of E. chaffeensis with 94.7% and 99.2% identity, respectively. Ehrlichia sp. HF and Candidatus Neoehrlichia mikurensis were also detected in I. ovatus. Furthermore, Ehrlichia sp. HF was isolated from laboratory mice that were intraperitoneal inoculated with I. ovatus tick homogenate. Some ehrlichial agents detected in Ixodes ticks might be a previously unknown Ehrlichia species. In this study, Candidatus N. mikurensis was detected in I. ovatus ticks. Because I. ovatus is distributed widely and cases of its tick bite in humans are ubiquitously reported in Japan, there is a potential for ehrlichiosis to be endemic to Japan, necessitating intensive surveillance of this infectious disease.

Spotted Fever Group Rickettsiae in Inner Mongolia, China, 2015-2016.

We found Rickettsia raoultii infection in 6/261 brucellosis-negative patients with fever of unknown origin in brucellosis-endemic Inner Mongolia, China. We further identified Hyalomma asiaticum ticks associated with R. raoultii, H. marginatum ticks associated with R. aeschlimannii, and Dermacentor nuttalli ticks associated with both rickettsiae species in the autonomous region.

Binding of Catechins to Staphylococcal Enterotoxin A.

Staphylococcal enterotoxin A (SEA) is a toxin protein, and is the most common cause of staphylococcal food poisoning. Polyphenols, such as catechins, are known to interact with proteins. In this study, we investigated the binding of catechins to SEA using SPR (Biacore), Fourier transform infrared spectroscopy (FT-IR), isothermal titration calorimetry (ITC), and protein-ligand docking. We found that (−)-epigallocatechin gallate (EGCG) could strongly bind to SEA. According to thermodynamic parameters, a negative ΔG indicated that the interaction between EGCG and SEA was spontaneous, and the electrostatic force accompanied by hydrophobic binding forces may play a major role in the binding. Data from Western blot analysis and docking simulation suggest that the hydroxyl group at position 3 of the galloyl group in the catechin structure was responsible for binding affinity with the Y91 of the A-6 region of SEA active sites. Our results provide further understanding of the binding interactions between catechins and SEA, and the inhibition of toxin activities by catechins.

Evaluation of Diagnostic Assay for Rickettsioses Using Duplex Real-Time PCR in Multiple Laboratories in Japan.

Tsutsugamushi disease and Japanese spotted fever are representative rickettsioses in Japan, and are caused by infection with Orientia tsutsugamushi and Rickettsia japonica, respectively. For molecular-based diagnosis, conventional PCR assays, which independently amplify respective rickettsial DNA, are usually used; however, this approach is time-consuming. Here, we describe a new duplex real-time PCR assay for the simultaneous detection of O. tsutsugamushi and spotted fever group rickettsiae, and its evaluation using several PCR conditions in 6 public health laboratories. The detection limit of the assay was estimated to be 102 copies and the sensitivity was almost identical to that of 3 conventional PCR methods. A total of 317 febrile patients were selected as clinically suspected or confirmed cases of rickettsioses. The detection efficiency of this assay for O. tsutsugamushi from blood or skin (eschar) specimens appeared to be almost the same as that of the conventional PCR method, even when performed in different laboratories, whereas the efficiency for spotted fever group rickettsiae tended to be higher than that of the 2 traditional double PCR assays. Our duplex real-time PCR is thus a powerful tool for the rapid diagnosis of rickettsioses, especially at the acute stage of infection.

Case of Human Infection with Anaplasma phagocytophilum in Inner Mongolia, China.

Anaplasma phagocytophilum is an obligate intracellular bacterium that causes febrile illness in humans and livestock. A 49-year-old woman was suffering from feverish symptoms, fatigue, arthralgia, general body pain, and anorexia for 2 weeks. Later, she visited the Bayannur Centers for Disease Control and Prevention Hospital in Inner Mongolia, China. Molecular-based diagnostic analysis of the patient's blood revealed that A. phagocytophilum p44 DNA was positive, but Brucella omp31, spotted fever group Rickettsia gltA, Orientia tsutsugamushi 16S rDNA, and Ehrlichia p28 were negative. The amino acid sequences of 9 A. phagocytophilum p44 clones obtained from the patient shared 44-100% similarity among them and were closely related to those of previously identified p44 clones from Canis familiaris (accession no. KJV64194) and from Ixodes persulcatus tick (no. BAN28309). Serological tests using the patient's serum showed that immunoglobulin M (IgM) and IgG titers to A. phagocytophilum antigens were 160 and 20, respectively, determined using indirect immunofluorescence assay, and the reaction to recombinant P44 proteins (rP44-1, rP44-18ES, and/or rP44-47) was confirmed using Western blot analysis. Thus, the results obtained in this study strongly suggest that the patient was infected with A. phagocytophilum. To our knowledge, this is the first case of human anaplasmosis infection in the Inner Mongolia Autonomous Region.

Inhibitory effects of food additives derived from polyphenols on staphylococcal enterotoxin A production and biofilm formation by Staphylococcus aureus.

In this study, we examined the inhibitory effects of 14 food additives derived from polyphenol samples on staphylococcal enterotoxin A (SEA) production and biofilm formation by Staphylococcus aureus. Tannic acid AL (TA), Purephenon 50 W (PP) and Polyphenon 70A (POP) at 0.25 mg/mL and Gravinol®-N (GN), Blackcurrant polyphenol AC10 (BP), and Resveratrol-P5 (RT) at 1.0 mg/mL significantly decreased SEA production by S. aureus C-29 (p < 0.05). TA, GN, BP, and RT significantly inhibited the expression of the sea gene in S. aureus C-29 (p < 0.05), while suppression attempts by PP and POP proved unsuccessful. After result analysis, it can be derived that TA, GN, BP, and RT inhibit the production of SEA. Of the six samples, each one significantly inhibited biofilm formation (p < 0.05). Food additives derived from polyphenols have viability to be used as a means to inhibit the enterotoxin production and control the biofilm formation of foodborne pathogens.

Interaction between Various Apple Procyanidin and Staphylococcal Enterotoxin A and Their Inhibitory Effects on Toxin Activity.

In this study, we investigated the interaction between apple polyphenols (AP; mainly consisting of procyanidin (PC) from an apple) and staphylococcal enterotoxin A (SEA), and the inhibitory effects of AP on SEA activity. According to the degree of polymerization, in particularly highly polymerized PC (more than pentamer) strongly interacted with SEA. The binding affinity of AP with SEA molecules was determined using Biacore analysis. AP reacted with SEA immobilized on a Biacore sensor chip. After treatment with pepsin and pancreatin, to examine the changes of binding affinity of AP in intragastric conditions, AP maintained interaction with SEA. We examined whether AP inhibits the proliferation and interferon-γ (IFN-γ) production induced by SEA in mouse spleen cells. AP strongly inactivated the proliferation and IFN-γ production induced by SEA. These results suggest that AP, which has a higher degree of polymerization, inactivates stronger biological activity of SEA through interaction with SEA. Our studies are the first to demonstrate the relationship between the degree of polymerization of AP and the inhibitory effects on SEA activities.

Case Report: Clinical Features of a Case of Suspected Borrelia miyamotoi Disease in Hokkaido, Japan.

We herein report a case of suspected Borrelia miyamotoi disease in Hokkaido, Japan. The patient complained of lassitude, arthralgia, and high fever after a tick bite. Furthermore, at the time of consultation, the patient exhibited momentary loss of consciousness and low blood pressure. Laboratory tests revealed elevation of liver enzymes, thrombocytopenia, and increased C-reactive protein. Seroconversion to B. miyamotoi glycerophosphoryl diester phosphodiesterase antigen suggested the patient was infected with a relapsing fever group Borrelia species.