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Sequential evaluation of dogs naturally infected with Ehrlichia canis, Ehrlichia chaffeensis, Ehrlichia equi, Ehrlichia ewingii, or Bartonella vinsonii.

Historically, disease manifestations in dogs seroreactive to Ehrlichia canis antigens by indirect immunofluorescent antibody testing have been attributed to infection with either E. canis or Ehrlichia ewingii. A 1996 study by Dawson and colleagues provided PCR evidence that healthy dogs from southeastern Virginia could be naturally infected with Ehrlichia chaffeensis. This observation stimulated us to determine which Ehrlichia spp. infected sick dogs that were referred to our hospital from the same region. Based upon PCR amplification with species-specific primers, sick dogs seroreactive to E. canis antigens were determined to be infected with four Ehrlichia species: E. canis, E. chaffeensis, E. equi, and E. ewingii. Coinfection with three Ehrlichia species (E. canis, E. ewingii, and E. equi) was documented for one dog. An additional canine pathogen presumed to be tick transmitted, Bartonella vinsonii subsp. berkhoffii, was identified in 7 of 12 dogs. Importantly, our results indicate that in naturally infected dogs, E. chaffeensis can cause severe disease manifestations that are clinically and serologically indistinguishable from disease manifestations of E. canis or E. ewingii. In addition, our findings support the efficacy of doxycycline for treatment of E. canis, E. equi, and E. ewingii infections but indicate that, based upon the persistence of E. chaffeensis DNA for 1 year following treatment, E. chaffeensis infection in dogs may be more refractory to doxycycline treatment. Undetected coinfection with Bartonella may also complicate the evaluation of treatment efficacy while resulting in disease manifestations that mimic ehrlichiosis.

Animals↗

Ehrlichial proliferation and acute hepatocellular necrosis in immunocompetent mice experimentally infected with the HF strain of Ehrlichia, closely related to Ehrlichia chaffeensis.

Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis, is closely related to the HF strain of Ehrlichia isolated from ticks in Japan. In this study, BALB/c mice inoculated intraperitoneally with the HF strain developed severe illness and died at about day 9 post-inoculation. At necropsy, diffuse liver necrosis was evident. Ehrlichial microcolonies were observed in endothelial cells, monocytes and macrophages of the liver, bone marrow, spleen, thymus, and large and small intestine. Immunocompetent mice infected with the HF strain would provide a useful model for studying pathogenesis and immunity in acute and severe ehrlichiosis caused by E. chaffeensis and related Ehrlichia spp.

Acute Disease↗

Clinical and biological aspects of infection caused by Ehrlichia chaffeensis.

Ehrlichia chaffeensis is an obligatory intracellular bacterium that infects the monocyte-macrophage. E. chaffeensis, which is transmitted to humans by ticks primarily from infected deer, causes human monocytic ehrlichiosis, an acute febrile systemic illness. This paper reviews current knowledge of clinical and biological aspects of infections caused by E. chaffeensis.

Animals↗

Cloning and sequencing of the gene for a 120-kDa immunodominant protein of Ehrlichia chaffeensis.

Ehrlichia chaffeensis is the tick-borne, obligately intracellular bacterium that causes human monocytic ehrlichiosis. A 120-kDa protein is one of the immunodominant proteins of E. chaffeensis that stimulates production of specific antibodies in infected humans. A genomic library of E. chaffeensis was constructed in a lambda ZAP II phage vector, and a clone expressing the 120-kDa protein of E. chaffeensis was identified using canine anti-E. chaffeensis serum. DNA sequence analysis of the cloned 120-kDa protein gene of E. chaffeensis identified a 1884-bp open reading frame with an ehrlichial promoter. Five identical 240-bp tandem repeat units were identified in the 120-kDa protein gene of E. chaffeensis, comprising 60% of the entire gene. Aside from the first repeat unit, all the other repeat units are identical. In the first repeat unit there are four nucleotides that are different from the other repeats. Hydropathy analysis of the deduced amino-acid sequence demonstrated that the repeat domain contains highly hydrophilic segments. The 120-kDa protein should be evaluated for a role in stimulating protective immunity.

Amino Acid Sequence↗

Protein kinase A-mediated inhibition of gamma interferon-induced tyrosine phosphorylation of Janus kinases and latent cytoplasmic transcription factors in human monocytes by Ehrlichia chaffeensis.

Ehrlichia chaffeensis, an obligatory intracellular bacterium of monocytes or macrophages, is the etiologic agent of human monocytic ehrlichiosis. Our previous study showed that gamma interferon (IFN-gamma) added prior to or at early stage of infection inhibited infection of human monocytes with E. chaffeensis; however, after 24 h of infection, IFN-gamma had no antiehrlichial effect. To test whether ehrlichial infection disrupts Janus kinase (Jak) and signal transducer and activator of transcription (Stat) signaling induced by IFN-gamma, tyrosine phosphorylation of Stat1, Jak1, and Jak2 in E. chaffeensis-infected THP-1 cells was examined by immunoprecipitation followed by immunoblot analysis. Viable E. chaffeensis organisms blocked tyrosine phosphorylation of Stat1, Jak1, and Jak2 in response to IFN-gamma within 30 min of infection. Similar results were obtained with human peripheral blood monocytes infected with E. chaffeensis. Heat or proteinase K treatment but not periodate treatment of E. chaffeensis abrogated the inhibitory effect, suggesting that protein factor(s) of E. chaffeensis is responsible for the inhibition of IFN-gamma-induced tyrosine phosphorylation. Preincubation of E. chaffeensis with the Fab fragment of dog anti-E. chaffeensis immunoglobulin G also abrogated the inhibitory effect. On the other hand, monodansylcadaverine, which does not block binding but blocks internalization of ehrlichiae into macrophages, did not have any influence on the tyrosine phosphorylation. These results indicate that ehrlichial binding to host cells is sufficient to inhibit Stat1 tyrosine phosphorylation induced by IFN-gamma. Protein kinase A (PKA) activity in THP-1 cells increased approximately 25-fold within 30 min of infection with E. chaffeensis. In THP-1 cells pretreated with a PKA inhibitor, Rp isomer of adenosine 3',5'-cyclic phosphorothioate, E. chaffeensis-induced inhibition of Stat1 tyrosine phosphorylation was partially abrogated. These results suggest that E. chaffeensis blocks IFN-gamma-induced tyrosine phosphorylation of Jak and Stat through raising PKA activity in THP-1 cells, which may be an important survival mechanism of ehrlichiae within the host cell.

Bacterial Adhesion↗

Absence of tumor necrosis factor alpha, interleukin-6 (IL-6), and granulocyte-macrophage colony-stimulating factor expression but presence of IL-1beta, IL-8, and IL-10 expression in human monocytes exposed to viable or killed Ehrlichia chaffeensis.

Ehrlichia chaffeensis is a recently isolated minute gram-negative obligatory intracellular bacterium of monocytes/macrophages and is the etiologic agent of human monocytic ehrlichiosis. It is not known how macrophages respond when they encounter ehrlichiae in terms of cytokine production. In this study, we examined cytokine mRNA expression by incubating E. chaffeensis with THP-1 cells and performing competitive reverse transcription-PCR (RT-PCR). At 2 h postinfection, the levels of interleukin-1beta (IL-1beta), IL-8, and IL-10 mRNAs were significant but lower than those following Escherichia coli lipopolysaccharide (LPS) stimulation. Unlike the situation with E. coli LPS stimulation, however, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-alpha) mRNAs were not induced. Time course and dose-response studies confirmed the absence of IL-6, GM-CSF, and TNF-alpha mRNA induction with E. chaffeensis. Viable E. chaffeensis organisms were not required for IL-1beta IO, IL-8, and IL-10 mRNA induction, since heat-killed E. chaffeensis induced identical time course responses. IL-1beta, IL-8, and IL-10 mRNAs were detected for up to 21, 21, and 24 h postexposure with E. chaffeensis, respectively, which were shut off more rapidly than with LPS stimulation. Although heat treatment of E. chaffeensis had no effect, periodate treatment completely abolished the ability of E. chaffeensis to induce IL-1beta, IL-8, and IL-10 mRNAs. The capture enzyme-linked immunosorbent assay result corresponded with the RT-PCR results, showing that viable and heat-killed E. chaffeensis produced and secreted the same levels of IL-1beta and IL-8. IL-10 production was significantly reduced by heat treatment. Periodate-treated ehrlichiae did not induce production of any of the cytokines tested. Anti-CD14 monoclonal antibody and polymyxin B did not inhibit IL-1beta mRNA expression upon exposure to E. chaffeensis. The absence of TNF-alpha, IL-6, and GM-CSF mRNA induction may delay the development of a protective immune response, thereby allowing E. chaffeensis to set up residence in macrophages.

Cytokines↗

Identification of the antigenic constituents of Ehrlichia chaffeensis.

Ehrlichia chaffeensis, the novel etiologic agent of human ehrlichiosis in the United States, was first isolated in 1990 and reported in 1991. To analyze the antigenic components of E. chaffeensis, we cultivated these obligate intracellular bacteria in DH82 cells, purified the ehrlichiae by renografin density gradient centrifugation, and examined the antigens by Western immunoblotting. Rabbit and human antisera to E. chaffeensis revealed more than 20 bands ranging from 20 to 200 kD. The distinct 22-kD protein was heat labile. The rest of the major immunoreactive components were heat stable. The immunoblots of E. chaffeensis were highly similar when probed with antisera to E. chaffeensis, E. canis, and E. ewingii, indicating the close antigenic relationships among the three species. The 22-kD protein cross-reacted only with anti-E. canis serum. The antibody against E. sennetsu reacted strongly with the 66-, 64-, 55-, and 44-kD antigens of E. chaffeensis. The E. risticii antisera reacted strongly with the 55- and 44-kD bands but only faintly with the 66-kD band. The major immunoreactive antigens of E. chaffeensis (66, 55, and 44 kD) showed cross-reactions with all the different antisera tested. The results indicated that E. chaffeensis is antigenically most closely related to E. canis, is less closely related to E. ewingii, and is only distantly related to E. sennetsu and E. risticii.

Animals↗

Unique macrophage and tick cell-specific protein expression from the p28/p30-outer membrane protein multigene locus in Ehrlichia chaffeensis and Ehrlichia canis.

Ehrlichia chaffeensis and Ehrlichia canis are tick-transmitted rickettsial pathogens that cause human and canine monocytic ehrlichiosis respectively. We tested the hypothesis that these pathogens express unique proteins in response to their growth in vertebrate and tick host cells and that this differential expression is similar in closely related Ehrlichia species. Evaluation of nine E. chaffeensis isolates and one E. canis isolate demonstrated that protein expression was host cell-dependent. The differentially expressed proteins included those from the p28/30-Omp multigene locus. E. chaffeensis and E. canis proteins expressed in infected macrophages were primarily the products of the p28-Omp 19 and 20 genes or their orthologues. In cultured tick cells, E. canis expressed only the p30-10 protein, an orthologue of the E. chaffeensis p28-Omp 14 protein which is the only protein expressed by E. chaffeensis propagated in cultured tick cells. The expressed Omp proteins were post-translationally modified to generate multiple molecular forms. E. chaffeensis gene expression from the p28/30-Omp locus was similar in tick cell lines derived from both vector (Amblyomma americanum) and non-vector (Ixodes scapularis) ticks. Differential expression of proteins within the p28/p30-Omp locus may therefore be vital for adaptation of Ehrlichia species to their dual host life cycle.

Amino Acid Sequence↗

Infections with Ehrlichia chaffeensis and Ehrlichia ewingii in persons coinfected with human immunodeficiency virus.

The clinical course and laboratory evaluation of 21 patients coinfected with human immunodeficiency virus (HIV) and Ehrlichia chaffeensis or Ehrlichia ewingii are reviewed and summarized, including 13 cases of ehrlichiosis caused by E. chaffeensis, 4 caused by E. ewingii, and 4 caused by either E. chaffeensis or E. ewingii. Twenty patients were male, and the median CD4(+) T lymphocyte count was 137 cells/microL. Exposures to infecting ticks were linked to recreational pursuits, occupations, and peridomestic activities. For 8 patients, a diagnosis of ehrlichiosis was not considered until > or =4 days after presentation. Severe manifestations occurred more frequently among patients infected with E. chaffeensis than they did among patients infected with E. ewingii, and all 6 deaths were caused by E. chaffeensis. Ehrlichiosis may be a life-threatening illness in HIV-infected persons, and the influence of multiple factors, including recent changes in the epidemiology and medical management of HIV infection, may increase the frequency with which ehrlichioses occur in this patient cohort.

Adult↗

Infection rates of Amblyomma americanum and Dermacentor variabilis by Ehrlichia chaffeensis and Ehrlichia ewingii in southwest Missouri.

Both Ehrlichia chaffeensis and Ehrlichia ewingii are causative agents of human ehrlichiosis. Both pathogens are transmitted to humans through the bite of an infected lone star tick (Amblyomma americanum). Since Missouri has a high incidence of human monocytic ehrlichiosis, we investigated the prevalence of E. chaffeensis- and E. ewingii-infected A. americanum and Dermacentor variabilis (American dog tick) ticks to help assess the relative risk for humans exposed to these vectors. We used a nested polymerase chain reaction assay for the detection of ehrlichial DNA in the collected ticks. Infection rates for both ehrlichial species were calculated from the assay results for each of the tick species. E. chaffeensis was found to be present in 9.8% of adult A. americanum ticks (57 of 579) and 6.7% of D. variabilis ticks (eight of 120). E. ewingii DNA was present at an infection rate of 5.4% in adult A. americanum (31 of 579) and 3.3% of D. variabilis ticks (four of 120). A minimum infection rate for nymph pools of A. americanum was 1.7% for E. chaffeensis and 0.6% for E. ewingii.

Animals↗

A real-time combined polymerase chain reaction assay for the rapid detection and differentiation of Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia ewingii.

A rapid real-time polymerase chain reaction (PCR) assay capable of the simultaneous detection and differentiation of Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia ewingii was developed using the LightCyclertrade mark instrument (Roche Applied Sciences, Indianapolis, IN). The assay targets the operon groEL of the heat shock protein. Base pair mismatches in amplified DNA in regions of detection probe hybridization allowed organism differentiation by melting curve analysis. The analytical sensitivity was at least 10 copies per reaction. DNA extracts from 59 specimens previously confirmed positive for A. phagocytophilum (n = 37), E. chaffeensis (n = 19), or E. ewingii (n = 3) were used to evaluate the assay. All of the specimens positive for 1 of the 3 organisms by conventional PCR were likewise positive by the LightCycler method. Sensitivity and specificity were at least 100% compared with conventional PCR. This assay provides a rapid method for the detection and differentiation of the causative agents of human ehrlichiosis in the United States.

Anaplasma phagocytophilum↗

Glycosylation of homologous immunodominant proteins of Ehrlichia chaffeensis and Ehrlichia canis.

The glycoprotein genes of Ehrlichia chaffeensis (1,644 bp) and Ehrlichia canis (2,064 bp) encode proteins of 548 to 688 amino acids with predicted molecular masses of only 61 and 73 kDa but with electrophoretic mobilities of 120 kDa (P120) and 140 kDa (P140), respectively. The 120-kDa protein gene of E. chaffeensis contains four identical 240-bp tandem repeat units, and the 140-kDa protein gene of E. canis has 14 nearly identical, tandemly arranged 108-bp repeat units. Conserved serine-rich motifs identified in the repeat units of P120 and P140 were also found in the repeat units of the human granulocytotropic ehrlichiosis agent 130-kDa protein and of the fimbria-associated adhesin protein Fap1 of Streptococcus parasanguis. Nearly the entire (99%) E. chaffeensis P120 gene (1,616 bp), the 14-repeat region (78%) of the E. canis P140 gene (1,620 bp), and a 2-repeat region from the E. chaffeensis P120 gene (520 bp) were expressed in Escherichia coli. The recombinant proteins exhibited molecular masses ranging from 1.6 to 2 times larger than those predicted by the amino acid sequences. Antibodies against the recombinant proteins reacted with E. chaffeensis P120 and E. canis P140, respectively. Carbohydrate was detected on the E. chaffeensis and E. canis recombinant proteins, including the two-repeat polypeptide region of E. chaffeensis P120. A carbohydrate compositional analysis identified glucose, galactose, and xylose on the recombinant proteins. The presence of only one site for N-linked (Asn-Xaa-Ser/Thr) glycosylation, a lack of effect of N-glycosidase F, the presence of 70 and 126 Ser/Thr glycosylation sites in the repeat regions of P120 and P140, respectively, and a high molar ratio of carbohydrate to protein suggest that the glycans may be O linked.

Amino Acid Sequence↗

Western immunoblotting analysis of the antibody responses of patients with human monocytotropic ehrlichiosis to different strains of Ehrlichia chaffeensis and Ehrlichia canis.

In order to evaluate the relative sensitivity of the detection of antibodies against various antigenic proteins of Ehrlichia chaffeensis for the diagnosis of the emerging infectious disease human monocytotropic ehrlichiosis, Western immunoblotting was performed with 27 serum samples from convalescent patients with antibodies, as demonstrated by indirect immunofluorescence assay. Among 22 patients with antibodies reactive with the 120-kDa protein, 15 showed reactivity with the 29/28-kDa protein(s) and the proteins in the 44- to 88-kDa range. Two of the serum samples with this pattern reacted with the 29/28-kDa protein(s) of only the 91HE17 strain, and one sample reacted with only that of the Arkansas strain, indicating that the antibodies were stimulated by strain-specific epitopes. Overall, antibodies to the 29/28-kDa protein(s) were detected in only 16 patients' sera, suggesting that this protein is less sensitive than the 120-kDa protein. Two of 12 serum samples from healthy blood donors had antibodies reactive with the 120-kDa protein; one of these samples reacted also with the 29/28-kDa protein(s) of Ehrlichia canis, suggesting that unrecognized ehrlichial infection might have occurred, including human infection with E. canis. A high correlation between reactivity with the 120-kDa protein by Western immunoblotting and the recombinant 120-kDa protein by dot blot supports the potential usefulness of this recombinant antigen in diagnostic serology.

Adult↗

Genetic diversity of the 28-kilodalton outer membrane protein gene in human isolates of Ehrlichia chaffeensis.

The Ehrlichia chaffeensis 28-kDa outer membrane protein (p28) gene was sequenced completely by genomic walking with adapter PCR. The DNA sequence of the p28 gene was nearly identical to the previously reported sequence (N. Ohashi, N. Zhi, Y. Zhang, and Y. Rikihisa, Infect. Immun. 66:132-139, 1998), but analysis of a further 75 bp on the 5' end of the gene revealed DNA that encoded a 25-amino-acid signal sequence. The leader sequence was removed from the N terminus of a 30-kDa precursor to generate the mature p28 protein. A monoclonal antibody (MAb), 1A9, recognizing four outer membrane proteins of E. chaffeensis (Arkansas strain) including the 25-, 26-, 27-, and 29-kDa proteins (X.-J. Yu, P. Brouqui, J. S. Dumler, and D. Raoult, J. Clin. Microbiol. 31:3284-3288, 1993) reacted with the recombinant p28 protein. This result indicated that the four proteins recognized by MAb 1A9 were encoded by the multiple genes of the 28-kDa protein family. DNA sequence alignment analysis revealed divergence of p28 among all five human isolates of E. chaffeensis. The E. chaffeensis strains could be divided into three genetic groups on the basis of the p28 gene. The first group consisted of the Sapulpa and St. Vincent strains. They had predicted amino acid sequences identical to each other. The second group contained strain 91HE17 and strain Jax, which only showed 0.4% divergence from each other. The third group contained the Arkansas strain only. The amino acid sequences of p28 differed by 11% between the first two groups, by 13.3% between the first and third groups, and by 13.1% between the second and third groups. The presence of antigenic variants of p28 among the strains of E. chaffeensis and the presence of multiple copies of heterogeneous genes suggest a possible mechanism by which E. chaffeensis might evade the host immune defenses. Whether or not immunization with the p28 of one strain of E. chaffeensis would confer cross-protection against other strains needs to be investigated.

Amino Acid Sequence↗

Hepatic pathology in human monocytic ehrlichiosis. Ehrlichia chaffeensis infection.

Ehrlichia chaffeensis causes human monocytic ehrlichiosis (HME) that usually includes fever, myalgias, and pancytopenia and, in 80% to 90% of patients, elevations in serum transaminase levels. Thus, the pathology of liver injury was studied in liver tissues from 7 patients with laboratory-confirmed HME. H&E and immunohistochemical stains for E chaffeensis and leukocyte markers were examined. Scattered lobular lymphohistiocytic foci and diffuse lymphohistiocytic infiltration and Kupffer cell hyperplasia with increased phagocytosis frequently were present. Various degrees of liver cell injury and death were observed. Cholestasis was evident in 6 cases, sometimes with bile duct epithelial injury. Rare to abundant E chaffeensis-infected mononuclear cells infiltrating lobules or portal regions or in Kupffer cells were observed in 5 patients. The inflammation was out of proportion to the infection in 6 cases. In the absence of infected hepatocytes or biliary epithelial cells, these findings suggest that host inflammatory or immune responses contribute to the liver injury seen in HME.

Ehrlichia chaffeensis↗

Development of a p28-based PCR assay for Ehrlichia chaffeensis.

Detection of Ehrlichia chaffeensis is necessary to study interactions between the parasite and its vertebrate and invertebrate hosts. The purpose of this study was to develop a sensitive, specific PCR assay for E. chaffeensis based on the outer membrane protein gene, p28. Candidate primer sets were identified and ranked based on annealing scores, similarities to three major p28 sequence clusters, dissimilarity to E. canis p30, an ortholog of p28, and the proximities of flanking primer sequences for nested PCR. The relative sensitivities of five optimized single-step and two nested PCR assays were determined, and the most sensitive assay was found to be a single-step PCR that was as much as 1000-fold more sensitive than a standard 16S rDNA-based nested PCR assay. This p28-based PCR assay amplified the target amplicon from isolates representative of all three major clusters of known p28 sequences, and this assay did not amplify template prepared from either of the two species most closely related to E. chaffeensis, E. canis and E. muris. These results indicate that this sensitive, specific and isolate-universal single-step PCR assay will be a useful tool in characterizing the transmission of this important zoonotic pathogen.

Bacterial Outer Membrane Proteins↗

Detection of Ehrlichia chaffeensis in Brazilian marsh deer (Blastocerus dichotomus).

Ehrlichia chaffeensis was detected for the first time in blood samples from Brazilian marsh deers (Blastocerus dichotomus) captured in the marshes of Parana River in Southeast Brazil in 1998. Seven EDTA-blood samples from deers were analyzed by PCR and nested PCR for presence of Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia canis, Neoriickettsia risticii, Anaplasma phagocytophilum and Anaplasma marginale. Three samples showed positive reactions for E. chaffeensis and Anaplasma marginale. None contained detectable A. phagocytophilum, E. ewingii, E. canis or Neorickettsia risticii DNA. In Brazil, the wild marsh deer may be a natural reservoir of the agents that cause human monocytotropic ehrlichiosis and ruminant erythrocytic anaplasmosis.

Anaplasma marginale↗

Ultrastructural variation of cultured Ehrlichia chaffeensis.

The ultrastructure of Ehrlichia chaffeensis (Arkansas strain) was studied in non-irradiated and irradiated monolayers of mouse embryo, Vero, BGM and L929 cells, and in non-irradiated DH82 cells. Within the intracellular parasitophorous vacuoles (morulae), two types of ehrlichial cells were found regularly--those with uniformly dispersed nucleoid filaments and ribosomes (dense-cored cells), which represent the normal life cycle of ehrlichiae. In addition, large reticulate cells were observed, forming long projections of the cell wall, protrusions of cytoplasmic membrane into the periplasmic space, or budding of protoplast fragments (minute forms) into the periplasmic space. Ehrlichiae with abnormalities of protoplast fission were found, apparently leading to formation of giant, multilobular or elongated rod-like ehrlichiae. Morulae were usually surrounded by cisterns of granular endoplasmic reticulum and mitochondria and often contained vesicles, long tubules 25nm in diameter, probably originating from the ehrlichial cell wall, and fibrillar ehrlichial antigen apparently shed from the surface of the cell wall. Some cells contained, in addition to normal morulae, a whole morula that had become dense and contained degenerating ehrlichiae. These results indicate that as well as normal growth and reproduction, ehrlichiae exhibit pathological events: they can be remarkably damaged inside the host cell vacuoles, presumably phagolysosomes, or enter a process morphologically similar to bacterial L-transformation.

Animals↗