Anaplasmataceae in wild rodents and roe deer from Trento Province (northern Italy).
Explore the source record for details and available documents.
Anaplasmataceae↗
Biomedical subjects
C Bandi
Publications and source records attributed to C Bandi.
Widespread distribution and high prevalence of an alpha-proteobacterial symbiont in the tick Ixodes ricinus.
The tick Ixodes ricinus is responsible for the transmission of a number of bacterial, protozoan and viral diseases to humans and animals in Europe and Northern Africa. Female I. ricinus from England, Switzerland and Italy have been found to harbour an intracellular alpha-proteobacterium, designated IricES1, within the cells of the ovary. IricES1 is the only prokaryote known to exist within the mitochondria of any animal or multicellular organism. To further examine the distribution, prevalence and mode of transmission of IricES1, we performed polymerase chain reaction screening of I. ricinus adults from 12 countries across its geographic distribution, including tick colonies that have been maintained in the laboratory for varying periods of time. IricES1 was detected in 100% of field-collected female ticks from all countries examined (n = 128), while 44% of males were found to be infected (n = 108). Those males that are infected appear to harbour fewer bacteria than females. Sequencing of fragments of the 16S rRNA and gyrB genes revealed very low nucleotide diversity among various populations of IricES1. Transmission of IricES1 from engorged adult females to eggs was found to be 100% (n = 31). In tick colonies that had been maintained in the laboratory for several years, a relatively low prevalence was found in females (32%; n = 25). To our knowledge, IricES1 is the most widespread and highly prevalent of any tick-associated symbiont.
Alphaproteobacteria↗
Phylogeny of Wolbachia pipientis based on gltA, groEL and ftsZ gene sequences: clustering of arthropod and nematode symbionts in the F supergroup, and evidence for further diversity in the Wolbachia tree.
Current phylogenies of the intracellular bacteria belonging to the genus Wolbachia identify six major clades (A-F), termed 'supergroups', but the branching order of these supergroups remains unresolved. Supergroups A, B and E include most of the wolbachiae found thus far in arthropods, while supergroups C and D include most of those found in filarial nematodes. Members of supergroup F have been found in arthropods (i.e. termites), and have previously been detected in the nematode Mansonella ozzardi, a causative agent of human filariasis. To resolve the phylogenetic positions of Wolbachia from Mansonella spp., and other novel strains from the flea Ctenocephalides felis and the filarial nematode Dipetalonema gracile, the authors generated new DNA sequences of the Wolbachia genes encoding citrate synthase (gltA), heat-shock protein 60 (groEL), and the cell division protein ftsZ. Phylogenetic analysis confirmed the designation of Wolbachia from Mansonella spp. as a member of the F supergroup. In addition, it was found that divergent lineages from Dip. gracile and Cte. felis lack any clear affiliation with known supergroups, indicating further genetic diversity within the Wolbachia genus. Finally, although the data generated did not permit clear resolution of the root of the global Wolbachia tree, the results suggest that the transfer of Wolbachia spp. from arthropods to nematodes (or vice versa) probably occurred more than once.
Animals↗
Molecular characterisation of a field strain of bubaline herpesvirus isolated from buffaloes (Bubalus bubalis) after pharmacological reactivation.
Two healthy buffaloes (Bubalus bubalis) in a herd which had not been vaccinated against infectious bovine rhinotracheitis (IBR), were selected for their seropositivity for anti-bovine herpesvirus type 1 (BoHV-1) glycoprotein E antibodies, and injected intramuscularly daily with dexamethasone for five consecutive days (day 1 to day 5) to reactivate any latent herpesvirus. Blood samples and nasal and vaginal swabs were collected daily from day 5 to day 15 from each buffalo for virological examination. All the vaginal swabs and blood samples were negative, but 13 of the 22 nasal swabs were positive; a cytopathic effect was observed in primary cultures of bovine fetal lung cells, and the viral isolates were identified as a herpesvirus by PCR. The viral strains were characterised by the sequence analysis of the genes coding for glycoproteins D and B, and the gene sequences were then used for phylogenetic analysis. The isolates from both buffaloes appeared identical at the level of the two genes, and were more closely related to bovine herpesvirus type 5 than to BoHV-1.
Amino Acid Sequence↗
Sexual transmission of a nematode parasite of Wood Mice (Apodemus sylvaticus)?
Sexual transmission occurs commonly in microparasites such as viruses and bacteria, but this is an unusual transmission route for macroparasites. Here we present evidence which suggests that a nematode parasite of Wood Mice (Apodemus sylvaticus) may be sexually transmitted and we have classified the nematode using molecular data. Wood Mice were collected annually in the course of work on their reproductive physiology. Larval nematodes were found in the epididymides of 19.6% of males. It seems likely that they would be transmitted to females at ejaculation. To identify these larval nematodes, which we were unable to do using morphological features, we sequenced the 18S rDNA. Sequence comparisons with the molecular phylogeny of Blaxter et al. (1998) demonstrated that they were bursate nematodes (Order Strongylida). The relationships between strongylid taxa were poorly resolved by 18S rDNA. However, both distance and parsimony analyses grouped the nematode with the superfamily Metastrongylidea in a clade containing Filaroides and Angiostrongylus sp. Importantly, the sequences were distinct from those of Heligmosomoides polygyrus and Angiostrongylus dujardini, two common strongylid nematodes of Apodemus. We were therefore unable positively to identify these worms by matching their sequences with those from morphologically identifiable adult strongylid nematodes infecting Apodemus. These results demonstrate that an as yet unidentified strongylid is quite commonly found in large numbers in the male reproductive tract of Wood Mice. Further work is required to understand the biology and transmission dynamics of this interesting system.
Animals↗
Clinical, cytological and molecular evidence of Mesocestoides sp. infection in a dog from Italy.
A 12-year-old, 13 kg, mixed-breed male dog was referred for anorexia and depression. The dog showed discomfort on abdominal palpation. Abdominal ultrasound examination revealed multiple, small, round anechoic cystic structures. Cystic fluid obtained with fine needle aspiration contained several 2-4 mm white motile flecks. Microscopic examination of the fluid revealed numerous irregularly shaped organisms measuring several hundred microns to 3 mm, the morphology of which was suggestive of intact and fragmented acephalic metacestodes of the genus Mesocestoides sp. Molecular analysis confirmed that the peritoneal infection was caused by Mesocestoides sp.
Animals↗
[Emerging rickettsioses].
Ticks are known to carry and transmit a number of microbial agents that cause diseases in humans and animals. Among these are members of the order Rickettsiales (alpha-proteobacteria), which include the genera Rickettsia and Ehrlichia. The most common and well-known Rickettsial human disease in Europe is Mediterranean Spotted Fever (MSF), caused by Rickettsia conorii. In recent years, a number of new Rickettsia species have been discovered in Europe, some of which have been shown to be pathogenic to humans. These discoveries have been facilitated by use of sequence-based molecular identification techniques. In Italy, it is generally believed that R. conorii is the only Rickettsia species present, and clinical tests for MSF rely on antigens raised against this bacterium. We are currently undertaking a molecular screening study of Rickettsiales-bacteria in ticks from various regions of Italy, to check for the potential presence of species from this order recently discovered in other parts of Europe. So far, we have identified a number of additional species in ticks collected from northern, central and southern regions. These include the known pathogens R. helvetica and R. slovaca as well as two species which may or may not be of medical relevance: R. monacensis and R. sp. IRS4. As a part of this survey, we have identified a novel alphaproteobacterium from the medically important tick Ixodes ricinus. This bacterium, tentatively named IricES1, has the unusual property of existing within the mitochondria, as well as the cytoplasm, of ovarian cells. To our knowledge, this is the only known example of a bacterium that is able to enter the mitochondria of animals. Our recently published electron microscopic data indicates that the bacterium enters mitochondria between the inner and outer membranes, and then proceeds to consume the inner mitochondrial matrix. We will present further data on this bacterium, including: 1) its phylogenetic position based on various molecular sequences, 2) its localization within the tick based on in situ hybridization; 3) its distribution among tick populations in Europe; 4) preliminary data on attempts at culturing this bacterium in a variety of cell types. Possible interactions between the bacterium and its host will be discussed. Ticks are known to carry and transmit a number of microbial agents that cause diseases in humans and animals. Among these are members of the order Rickettsiales (alpha-proteobacteria), which include the genera Rickettsia and Ehrlichia. The most common and well-known Rickettsial human disease in Europe is Mediterranean Spotted Fever (MSF), caused by Rickettsia conorii. In recent years, a number of new Rickettsia species have been discovered in Europe, some of which have been shown to be pathogenic to humans. These discoveries have been facilitated by use of sequence-based molecular identification techniques. In Italy, it is generally believed that R. conorii is the only Rickettsia species present, and clinical tests for MSF rely on antigens raised against this bacterium. We are currently undertaking a molecular screening study of Rickettsiales-bacteria in ticks from various regions of Italy, to check for the potential presence of species from this order recently discovered in other parts of Europe. So far, we have identified a number of additional species in ticks collected from northern, central and southern regions. These include the known pathogens R. helvetica and R. slovaca as well as two species which may or may not be of medical relevance: R. monacensis and R. sp. IRS4. As a part of this survey, we have identified a novel alphaproteobacterium from the medically important tick Ixodes ricinus. This bacterium, tentatively named IricES1, has the unusual property of existing within the mitochondria, as well as the cytoplasm, of ovarian cells. To our knowledge, this is the only known example of a bacterium that is able to enter the mitochondria of animals. Our recently published electron microscopic data indicates that the bacterium enters mitochondria between the inner and outer membranes, and then proceeds to consume the inner mitochondrial matrix. We will present further data on this bacterium, including: 1) its phylogenetic position based on various molecular sequences, 2) its localization within the tick based on in situ hybridization; 3) its distribution among tick populations in Europe; 4) preliminary data on attempts at culturing this bacterium in a variety of cell types. Possible interactions between the bacterium and its host will be discussed.
Animals↗
[Quantitative PCR in the diagnosis of Leishmania].
Polymerase chain reaction (PCR) is a sensitive and rapid method for the diagnosis of canine Leishmania infection and can be performed on a variety of biological samples, including peripheral blood, lymph node, bone marrow and skin. Standard PCR requires electrophoretic analysis of the amplification products and is usually not suitable for quantification of the template DNA (unless competitor-based or other methods are developed), being of reduced usefulness when accurate monitoring of target DNA is required. Quantitative real-time PCR allows the continuous monitoring of the accumulation of PCR products during the amplification reaction. This allows the identification of the cycle of near-logarithmic PCR product generation (threshold cycle) and, by inference, the relative quantification of the template DNA present at the start of the reaction. Since the amplification product are monitored in "real-time" as they form cycle-by-cycle, no post-amplification handling is required. The absolute quantification is performed according either to an internal standard co-amplified with the sample DNA, or to an external standard curve obtained by parallel amplification of serial known concentrations of a reference DNA sequence. From the quantification of the template DNA, an estimation of the relative load of parasites in the different samples can be obtained. The advantages compared to standard and semi-quantitative PCR techniques are reduction of the assay's time and contamination risks, and improved sensitivity. As for standard PCR, the minimal components of the quantitative PCR reaction mixture are the DNA target of the amplification, an oligonucleotide primer pair flanking the target sequence, a suitable DNA polymerase, deoxynucleotides, buffer and salts. Different technologies have been set up for the monitoring of amplification products, generally based on the use of fluorescent probes. For instance, SYBR Green technology is a non-specific detection system based on a fluorescent dsDNA intercalator and it is applicable to all potential targets. TaqMan technology is more specific since performs the direct assessment of the amount of amplified DNA using a fluorescent probe specific for the target sequence flanked by the primer pair. This probe is an oligonucleotide labelled with a reporter dye (fluorescent) and a quencher (which absorbs the fluorescent signal generated by the reporter). The thermic protocol of amplification allows the binding of the fluorescent probe to the target sequence before the binding of the primers and the starting of the polymerization by Taq polymerase. During polymerization, 5'-3' exonuclease activity of Taq polymerase digests the probe and in this way the reporter dye is released from the probe and a fluorescent signal is detected. The intensity of the signal accumulates at the end of each cycle and is related to the amount of the amplification product. In recent years, quantitative PCR methods based either on SYBR Green or TaqMan technology have been set up for the quantification of Leishmania in mouse liver, mouse skin and human peripheral blood, targeting either single-copy chromosomal or multi-copy minicircle sequences with high sensitivity and reproducibility. In particular, real-time PCR seems to be a reliable, rapid and noninvasive method for the diagnosis and follow up of visceral leishmaniasis in humans. At present, the application of real-time PCR for research and clinical diagnosis of Leishmania infection in dogs is still foreseable. As for standard PCR, the high sensitivity of real-time PCR could allow the use of blood sampling that is less invasive and easily performed for monitoring the status of the dogs. The development of a real-time PCR assay for Leishmania infantum infection in dogs could support the standard and optimized serological and PCR methods currenly in use for the diagnosis and follow-up of canine leishmaniasis, and perhaps prediction of recurrences associated with tissue loads of residual pathogens after treatment. At this regard, a TaqMan Real Time PCR method developed for the quantification of Leishmania infantum minicircle DNA in peripheral blood of naturally infected dogs sampled before and at different time points after the beginning of a standard antileishmanial therapy will be illustrated.
Animals↗
Sequencing of the complete gene coding for the GroEL of the Wolbachia of Dirofilaria immitis and expression and purification of the recombinant protein.
Wolbachia are intracellular bacteria that infect arthropods and filarial nematodes. These bacteria play an important role in the immunology and pathogenesis of filarial diseases through their proteins and, possibly, other molecules. GroEL is a constitutively expressed bacterial protein; it is highly conserved among bacteria and is involved in the correct folding of newly synthesized proteins. Here we report the production of recombinant GroEL from the Wolbachia of Dirofilaria immitis. Our goal is to test the hypothesis that GroEL is involved in the immunopathology of filariases. The complete groel gene was PCR-amplified, sequenced and cloned into an expression vector. The recombinant GroEL was purified by affinity chromatography by using high-performance liquid chromatography (HPLC).
Animals↗
Immunological role of the endosymbionts of Dirofilaria immitis: the Wolbachia surface protein activates canine neutrophils with production of IL-8.
Filarial nematodes, including Dirofilaria immitis and D. repens, harbour intracellular bacteria belonging to the genus Wolbachia. These bacteria have been implicated in the pathogenesis of filarial diseases, possibly through their endotoxins. Recent studies have shown that a major surface protein of Wolbachia (WSP) induces a specific IgG response in hosts infected by D. immitis. WSP from the Wolbachia of D. immitis was produced in recombinant form. The purified protein was used in stimulation assays on canine neutrophils. The assays performed using a modified Boyden chamber showed that WSP stimulates neutrophil chemokinesis. In addition, RT-PCR revealed increased production of chemokine IL-8 by cells incubated with this protein. Neutrophils have been shown to play a major role in the pathogenesis of river blindness, and to accumulate in the nodules of onchocerciasis patients. In dogs infected by D. immitis, neutrophils accumulate in kidneys and in the wall of pulmonary arteries. As shown by our studies, Wolbachia could contribute to these inflammatory phenomena through its surface protein WSP, independently from its endotoxin component.
Animals↗
Immunoglobulin G antibodies against the endosymbionts of filarial nematodes (Wolbachia) in patients with pulmonary dirofilariasis.
The dog parasite Dirofilaria immitis can infect humans. Patients with pulmonary dirofilariasis were tested for immunoglobulin G (IgG) antibodies against the surface protein of Wolbachia, the bacterial endosymbiont of D. immitis. These patients showed significantly higher IgG titers than healthy individuals from areas in which D. immitis was endemic as well as areas in which it was not endemic. Titration of anti-Wolbachia surface protein IgG could become useful for diagnostic applications.
Animals↗
dnaA gene sequences from Wolbachia pipientis support subdivision into supergroups and provide no evidence for recombination in the lineages infecting nematodes.
Wolbachia pipientis is an intracellular bacterial endosymbiont of arthropods and filarial nematodes. Six main supergroups of W. pipientis have been described: supergroups A, B, E, and F encompass arthropod wolbachiae; supergroups C and D encompass nematode wolbachiae. The description of these six supergroups has been based on the analysis of only two genes (ftsZ and 16S rDNA) and before decisions are taken on the taxonomic status of the six supergroups, analysis of further genes is required. In addition, the branching order of the six supergroups is still unresolved. Sequence information from other genes is also needed to allow phylogenesis to be addressed through the analysis of a higher number of characters. Here we report sequences from a portion of the gene coding for the DNAA protein of W pipientis, generated from the endosymbionts of 22 host species. Phylogenies based on dnaA gene sequences are congruent with the existence of at least six supergroups of W pipientis. In addition, subtrees generated for nematode wolbachiae in supergroups C and D were compared to the trees based on the already available gene sequences (ftsZ, 16S rDNA and wsp). The congruence observed among the trees based on the different genes agrees with the hypothesis that recombination does not occur in nematode wolbachiae.
Animals↗
Tetracycline treatment and sex-ratio distortion: a role for Wolbachia in the moulting of filarial nematodes?
Filarial nematodes harbour intracellular bacteria of the genus Wolbachia. These bacteria are thought to be beneficial to the host nematode. Indeed, tetracycline treatments reduce the population of Wolbachia in filarial worms and have detrimental effects on the nematode. Even though various antibiotic-curing experiments have been performed on filariae, the actual role of Wolbachia in the biology of these nematodes is not yet clear. To address this issue, we designed a first experiment on a model filaria (Brugia pahangi), maintained in the gerbil (Meriones unguiculatus). In this experiment, timing of tetracycline treatment was set on the basis of the larval stage of the nematode. This first experiment showed that 2 weeks of treatment started after the L(4)-L(5) moult of males, but before the moult of females, led to significant sex-ratio distortion of the nematodes. We thus hypothesised that tetracycline interferes with the moult in B. pahangi. To test this hypothesis, we designed a second experiment in which antibiotic treatments were started (1). before the moult of both sexes, (2). after the moult of males but before the moult of females, or (3). after the moult of both sexes. Treatment 1 determined a reduction of worm recovery with no sex bias. Treatment 2 led to a male-biased sex-ratio. Treatment 3 had no effect on either worm recovery or sex-ratio. These results thus support the hypothesis that tetracycline treatment interferes with the L(4)-L(5) moult of B. pahangi. The nematodes recovered from the treated and control animals were examined for the presence of Wolbachia using both immunohistochemistry and real-time PCR. In general, nematodes from treated animals showed a dramatic reduction in Wolbachia content. In one group, Wolbachia depletion, as observed at the end of the treatment, was followed by a rebound to 'normal' values 160 days later. Prospects for antifilarial therapy using Wolbachia-targeted tetracycline treatments should thus take into account the possibility of Wolbachia rebound.
Animals↗
Does fertilization in the filarial nematode Dirofilaria immitis occur through endocytosis of spermatozoa?
Information on the ultrastructural details of fertilization in filarial nematodes are still unavailable. Here we report new data on this process in Dirofilaria immits, the heartworm of dogs and cats. Electron microscopy allowed us to observe oocytes engulfing spermatozoa through an endocytosis-like process. We also observed spermatozoa inside the oocytes which still possessed their plasma membrane and which were clearly enveloped by a further membrane, likely derived from the endocytosis process. At this stage, at the interface between the sperm membrane and the endocytotic membrane (vacuolar space), we observed flocculent material in the proximity of the membranous organelles (MOs) of the sperm. In the proximity of the MOs, we also observed the enlargement of the vacuolar space. Other images showed the dissolution of the sperm membrane, and the release of nuclear masses and organelles in the egg cytoplasm. We did not observe the fusion of lysosomes to the endocytotic vacuoles. In addition, the lysis of the sperm organelles has never been observed inside the vacuoles containing the whole sperm. Thus we suggest that the degradation of the endocytotic and sperm plasma membranes is determined by material released by the MOs. Since we did not observe the entry of sperm into the oocytes by other mechanisms, we also suggest that endocytosis is the normal process used by the spermatozoon to get into the egg cytoplasm in D. immitis. Finally, during our observations of the seminal receptacle we did not observe any structure in the spermatozoa which could be interpreted as an intracellular bacterium. This is consistent with previous results indicating that the bacterium Wolbachia in filarial nematodes is not transmitted through the sperm.
Animals↗
Distribution pattern of bovine viral diarrhoea virus strains in intensive cattle herds in Italy.
The genetic variation of bovine viral diarrhoea virus (BVDV) was studied by comparative nucleotide sequence analysis of 26 Italian field strains collected during the period 1995-2000 in 18 cattle herds. A fragment within the 5'-untranslated region (UTR) was sequenced directly from gel-purified products obtained by reverse transcription polymerase chain reaction. BVDV-1b (n=14), -1c (n=1), -1d (n=1) and BVDV-2 (n=2) strains have been isolated. Most herds were infected by BVDV-1b. Pairwise similarity and cluster analysis of the remaining BVDV-1 isolates (n=8) did not provide a clear-cut assignation to defined BVDV-1 groups. This is the first time that a BVDV-2 isolation was reported in Italy. Among BVDV-2 reference strains, Italian BVDV-2 isolates showed the highest sequence similarity with the CD87 strain. Both BVDV-2 strains were isolated in two healthy animals from different herds. The 5'-UTR sequence of one of the two BVDV-2 strains was identical to a German BVDV field strain. Complete nucleotide homology was found only among BVDV strains isolated from the same herd, showing a herd-specific clustering. Moreover, 99.6% homology was observed between strains from herds linked by livestock trade. Despite the small number of BVDV isolates analysed, it revealed a high level of genetic diversity among Italian field BVDV strains.
5' Untranslated Regions↗
Wolbachia in filarial nematodes: evolutionary aspects and implications for the pathogenesis and treatment of filarial diseases.
The presence of intracellular bacteria in the body of various species of filarial nematodes, including important parasites such as Brugia malayi, Dirofilaria immitis, and Onchocerca volvulus, was observed as early as the mid-1970s. These bacteria were shown to be transovarially transmitted (from the female worm to the offspring) and to be present in significant amounts in the body of the nematode. As highlighted by their discoverers, the potential importance of these bacteria is fairly obvious: (1) bacteria-derived molecules should be considered as having an immunological and pathological role in filarial diseases; (2) the interaction between the bacteria and the filarial host deserves investigation, in view of the possibility that the bacteria are needed by the host nematode and could thus represent a target for therapy. Other authors, independently from the discovery of these intracellular bacteria, showed that the antibiotic tetracycline (which is well known for its efficacy on intracellular bacteria) had detrimental effects on two species of filarial nematodes (Brugia pahangi and Litomosoides sigmodontis). It is therefore surprising that for more than 20 years, no further investigations focused on the bacteria of filarial nematodes, nor on the anti-filarial properties of tetracycline. Recently, the bacteria of filarial nematodes have been independently "rediscovered" by research groups from the schools of Hamburg, Liverpool and Milan. These bacteria are now classified as Wolbachia, and the basic aspects of their phylogenetic history and relationship with the Wolbachia of arthropods have been reconstructed. In addition, their implications for the pathogenesis and treatment of filarial diseases have started to be uncovered. This paper, which is authored by representatives of the three European schools who reopened this research area, reviews our present knowledge of these fascinating microorganisms, highlighting the complexity of a symbiotic system which involves, in addition to the nematode and its bacterium, the vertebrate host.
Animals↗
Molecular identification of Wolbachia from the filarial nematode Mansonella ozzardi.
Mansonella ozzardi, a filarial parasite of humans in Latin America, has been shown to harbour intracellular bacteria not yet identified. Here we show that these bacteria, like those of other filarial nematodes, belong to the genus Wolbachia (alpha 2 Proteobacteria; Rickettsiales). Their unambiguous placement in the Wolbachia group was shown by 16S rDNA sequence analysis. However, the exact position of the Wolbachia from M. ozzardi relative to the other wolbachiae is not clear. Indeed, 16S rDNA sequence analysis places this bacterium at a deep branch in Wolbachia evolution. It is interesting that analysis of the 5S rDNA gene spacer of the nematode host also suggests that the genus Mansonella, together with the genus Loa, could represent a deep-branching lineage in filarial evolution.
Animals↗
Inherited microorganisms, sex-specific virulence and reproductive parasitism.
Parasites show an amazing repertoire of adaptations, highlighted by complex life cycles that allow both survival in the host and transmission among hosts. However, there is one heterogeneous group of microorganisms whose adaptations are perhaps even more surprising: parthenogenesis induction, feminization of genetic males, killing of male hosts and sperm-mediated sterilization of uninfected eggs. The common feature of these microorganisms is their mode of transmission: inheritance from mother to offspring. Here, we present an introduction to hereditary symbiosis, focusing on microsporidia and bacteria that manipulate host reproduction in arthropods (reproductive parasites). We also discuss the implications of one of these microorganisms, Wolbachia, for the control of arthropod pests and vectors and for the therapy of filarial diseases. Finally, we discuss whether some parasites of vertebrates might show sex-specific virulence.
Animals↗