Pathogenicity of genotype 2.1 classical swine fever virus isolated from Japan in 2019 in pigs.

Classical swine fever (CSF) re-emerged in Japan in 2018 for the first time in 26 years. The disease has been known to be caused by a moderately pathogenic virus, rather than the highly pathogenic virus that had occurred in the past. However, the underlying pathophysiology remains unknown. This study conducted an experimental challenge on specific pathogen-free (SPF) pigs in a naïve state for 2, 4, and 6 weeks and confirmed the disease state during each period by clinical observation, virus detection, and pathological necropsy. We revealed the pathological changes and distribution of pathogens and virus-specific antibodies at each period after virus challenge. These results were comprehensively analyzed and approximately 70% of the pigs recovered, especially at 4- and 6-week post-virus challenge. This study provides useful information for future countermeasures against CSF by clarifying the pathogenicity outcomes in unvaccinated pigs with moderately pathogenic genotype 2.1 virus.

Potent immunogenicity and neutralization of recombinant adeno-associated virus expressing the glycoprotein of severe fever with thrombocytopenia virus.

Severe fever with thrombocytopenia syndrome (SFTS) is an infectious disease caused by a tick-borne virus called severe fever with thrombocytopenia syndrome virus (SFTSV). In recent years, human infections through contact with ticks and through contact with the bodily fluids of infected dogs and cats have been reported; however, no vaccine is currently available. SFTSV has two glycoproteins (Gn and Gc) on its envelope, which are vaccine-target antigens involved in immunogenicity. In the present study, we constructed novel SFTS vaccine candidates using an adeno-associated virus (AAV) vector to transport the SFTSV glycoprotein genome. AAV vectors are widely used in gene therapy and their safety has been confirmed in clinical trials. Recently, AAV vectors have been used to develop influenza and SARS-CoV-2 vaccines. Two types of vaccines (AAV9-SFTSV Gn and AAV9-SFTSV Gc) carrying SFTSV Gn and Gc genes were produced. The expression of Gn and Gc proteins in HEK293T cells was confirmed by infection with vaccines. These vaccines were inoculated into mice, and the collected sera produced anti-SFTS antibodies. Furthermore, sera from AAV9-SFTSV Gn infected mice showed a potent neutralizing ability, similar to previously reported SFTS vaccine candidates that protected animals from SFTSV infection. These findings suggest that this vaccine is a promising candidate for a new SFTS vaccine.

Low potential of persistence and dissemination of antimicrobial-resistant Enterobacterales by wild nutria (Myocastor coypus) in a local river of Gifu Prefecture.

Environmental pollution caused by antimicrobial resistance is a global public health concern. To investigate the contribution of nutrias (Myocastor coypus) to the presence of extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales in the Ijira River, prevalence of ESBL-producing Enterobacterales in their feces was examined using deoxycholate-hydrogen sulfide-lactose agar containing cefotaxime. Additionally, the composition of the fecal microbiota of nutria was examined using DNA metabarcoding analysis of the 16S ribosomal RNA gene and compared with that of Amami rabbit, deer, fox, and raccoon dog. The absence of ESBL-producing Enterobacterales and substantially lower abundance of Enterobacterales was observed in the feces of nutrias than in those of other wild mammals. Our results suggest the low potential of antimicrobial-resistant Enterobacterales persistence and dissemination by nutria.

Diverse Molecular Mechanisms Underlying Microbe-Inducing Male Killing in the Moth Homona magnanima.

Male killing (MK) is a type of reproductive manipulation induced by microbes, where sons of infected mothers are killed during development. MK is a strategy that enhances the fitness of the microbes, and the underlying mechanisms and the process of their evolution have attracted substantial attention. Homona magnanima, a moth, harbors two embryonic MK bacteria, namely, Wolbachia (Alphaproteobacteria) and Spiroplasma (Mollicutes), and a larval MK virus, Osugoroshi virus (OGV; Partitiviridae). However, whether the three distantly related male killers employ similar or different mechanisms to accomplish MK remains unknown. Here, we clarified the differential effects of the three male killers on the sex-determination cascades and development of H. magnanima males. Reverse transcription-PCR demonstrated that Wolbachia and Spiroplasma, but not OGVs, disrupted the sex-determination cascade of males by inducing female-type splice variants of doublesex (dsx), a downstream regulator of the sex-determining gene cascade. We also found that MK microbes altered host transcriptomes in different manners; Wolbachia impaired the host dosage compensation system, whereas Spiroplasma and OGVs did not. Moreover, Wolbachia and Spiroplasma, but not OGVs, triggered abnormal apoptosis in male embryos. These findings suggest that distantly related microbes employ distinct machineries to kill males of the identical host species, which would be the outcome of the convergent evolution. IMPORTANCE Many microbes induce male killing (MK) in various insect species. However, it is not well understood whether microbes adopt similar or different MK mechanisms. This gap in our knowledge is partly because different insect models have been examined for each MK microbe. Here, we compared three taxonomically distinct male killers (i.e., Wolbachia, Spiroplasma, and a partiti-like virus) that infect the same host. We provided evidence that microbes can cause MK through distinct mechanisms that differ in the expression of genes involved in sex determination, dosage compensation, and apoptosis. These results imply independent evolutionary scenarios for the acquisition of their MK ability.

Isolation of extended-spectrum ß-lactamase-producing Escherichia coli from Japanese red fox (Vulpes vulpes japonica).

Antimicrobial resistance is a global concern requiring a one-health approach. Given wild animals can harbor antimicrobial-resistant bacteria (ARB), we investigated their presence in 11 fecal samples from wild animals using deoxycholate hydrogen sulfide lactose agar with or without cefotaxime (CTX, 1 mg/L). Thus, we isolated CTX-resistant Escherichia coli from two Japanese red fox fecal samples. One strain was O83:H42-ST1485-fimH58 CTX-M-55-producing E. coli carrying the genes aph(3″)-Ib, aph(3')-Ia, aph(6)-Id, mdf(A), sitABCD, sul2, tet(A), and tet(B), whereas the other was O25:H4-ST131-fimH30 CTX-M-14-producing E. coli carrying mdf(A) and sitABCD and showing fluoroquinolone resistance. Thus, the presence of extended-spectrum ß-lactamase producers in wild foxes suggests a spillover of ARB from human activities to these wild animals.

Establishment of an experimental model of normal dog bladder organoid using a three-dimensional culture method.

Dog bladder cancer (BC) is mostly muscle-invasive (MI) with poor prognosis, and its pathogenesis is close to human MIBC. Three-dimensional (3D) organoid culture ensures novel knowledge on cancer diseases including BC. Recently, we have established dog BC organoids (BCO) using their urine samples. BCO recapitulated the epithelial structures, characteristics, and drug sensitivity of BC-diseased dogs. However, organoids from dog normal bladder epithelium are not established yet. Therefore, the present study aimed to establish dog normal bladder organoids (NBO) for further understanding the pathogenesis of dog BC and human MIBC. The established NBO underwent various analyzes including cell marker expressions, histopathological structures, cancer-related gene expression patterns, and drug sensitivity. NBO could be produced non-invasively with a continuous culturing and recapitulated the structures and characteristics of the dog's normal bladder mucosal tissues. Different drug sensitivities were observed in each NBO. The analysis of RNA sequencing revealed that several novel genes were changed in NBO compared with BCO. NBO showed a higher expression of p53 and E-cadherin, but a lower expression of MDM2 and Twist1 compared with BCO. These results suggest that NBO could be a promising experimental 3D model for studying the developmental mechanisms of dog BC and human MIBC.

Validation of the usefulness of 26S rDNA D1/D2, internal transcribed spacer, and intergenic spacer 1 for molecular epidemiological analysis of Macrorhabdus ornithogaster.

Macrorhabdus ornithogaster (MO) is an infectious fungus that causes gastric damage in birds. In this study, we established nested and seminested polymerase chain reaction (PCR) methods that specifically amplify the domain D1/D2 region (D1/D2) of 26S ribosomal DNA (rDNA), internal transcribed spacer (ITS) of rDNA, and intergenic spacer (IGS) 1 region from avian feces. Phylogenetic analysis of MO collected from Japanese pet birds showed little genetic variation; analysis based on these regions did not distinguish between host species order, differences in MO shape, or host gastrointestinal symptoms. These regions were found to be unsuitable for molecular epidemiological studies of MO and further investigation into other genetic regions is required.

Establishment of Intestinal Organoid from Rousettus leschenaultii and the Susceptibility to Bat-Associated Viruses, SARS-CoV-2 and Pteropine Orthoreovirus.

Various pathogens, such as Ebola virus, Marburg virus, Nipah virus, Hendra virus, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and SARS-CoV-2, are threatening human health worldwide. The natural hosts of these pathogens are thought to be bats. The rousette bat, a megabat, is thought to be a natural reservoir of filoviruses, including Ebola and Marburg viruses. Additionally, the rousette bat showed a transient infection in the experimental inoculation of SARS-CoV-2. In the current study, we established and characterized intestinal organoids from Leschenault's rousette, Rousettus leschenaultii. The established organoids successfully recapitulated the characteristics of intestinal epithelial structure and morphology, and the appropriate supplements necessary for long-term stable culture were identified. The organoid showed susceptibility to Pteropine orthoreovirus (PRV) but not to SARS-CoV-2 in experimental inoculation. This is the first report of the establishment of an expandable organoid culture system of the rousette bat intestinal organoid and its sensitivity to bat-associated viruses, PRV and SARS-CoV-2. This organoid is a useful tool for the elucidation of tolerance mechanisms of the emerging rousette bat-associated viruses such as Ebola and Marburg virus.

African pygmy hedgehog adenovirus: Virus replication, virus-induced cytopathogenesis and activation of mitogen-activated protein kinase signaling pathways in infected MDCK cells.

We examined several aspects of African hedgehog adenovirus (AhAdv-1) that was isolated from an African pygmy hedgehog, including: replication kinetics of, virus-induced cytopathic effect (CPE), activation status of mitogen-activated protein kinase (MAPK) signaling pathways, and possible roles of these signaling pathways in virus replication and virus-induced CPE in MDCK cells. AhAdv-1 efficiently replicated and induced CPE in infected cells and caused accumulation of cleaved caspase-3 at 24 h post-infection (p.i.), suggesting apoptosis induction. Analysis of several intracellular signal transduction pathways, which are involved in apoptosis, showed activation of p38 MAPK, Akt and ERK1/2 pathways at 3 h p.i., and upregulation of phosphorylated SAPK/JNK at 24 h p.i. Although p38 MAPK inhibitor and SAPK/JNK inhibitor suppressed activation of the respective pathways in infected cells, they did not inhibit virus-induced CPE. Treatment of infected cells with inhibitor of the Akt pathway, the p38 pathway, the SAPK/JNK pathway or the ERK pathway revealed that inhibitors of p38 pathway inhibited viral replication by real-time PCR and TCID50 assay in infected MDCK cells, suggesting that AhAdv-1 uses p38 pathway for multiplication in infected cells.

Prevalence of antimicrobial resistance in bacteria isolated from Great Cormorants (Phalacrocorax carbo hanedae) in Japan.

Wild birds are recognized as disseminators of antimicrobial-resistant (AMR) bacteria into the environment. Here, we isolated AMR indicator bacteria from 198 Great Cormorant cloacal swabs collected in Shiga (n=90), Oita (n=52), Gifu (n=29), and Gunma (n=27) Prefectures, Japan, in 2018 and 2019. In total, 198 Aeromonas spp. and 194 Escherichia spp. were isolated, and their antimicrobial susceptibility was examined. Aeromonas spp. were resistant to colistin (8.6%), nalidixic acid (4%), and other antimicrobials (<2%), with 3.0% positivity for mcr-3. Escherichia spp. showed resistance to colistin (3.1%), ampicillin (2.6%), tetracycline (2.1%), and other antimicrobials (<2%). This study shows the presence of AMR bacteria in Great Cormorants, indicating that these birds potentially disseminate AMR bacteria.

Viral-derived DNA invasion and individual variation in an Indonesian population of large flying fox Pteropus vampyrus.

Here, we performed next-generation sequencing (NGS) on six large flying foxes (Pteropus vampyrus) collected in Indonesia. Seventy-five virus species in the liver tissue of each specimen were listed. Viral homologous sequences in the bat genome were identified from the listed viruses. This finding provides collateral evidence of viral endogenization into the host genome. We found that two of the six specimens bore partial sequences that were homologous to the plant pathogens Geminiviridae and Luteoviridae. These sequences were absent in the P. vampyrus chromosomal sequences. Hence, plant viral homologous sequences were localized to the hepatocytes as extrachromosomal DNA fragments. Therefore, this suggests that the bat is a potential carrier or vector of plant viruses. The present investigation on wild animals offered novel perspectives on viral invasion, variation, and host interaction.

Association between the blaCTX-M-14-harboring Escherichia coli Isolated from Weasels and Domestic Animals Reared on a University Campus.

Antimicrobial-resistant (AMR) bacteria affect human and animal health worldwide. Here, CTX-M-14-producing Escherichia coli isolates were isolated from Siberian weasels (Mustela sibirica) that were captured on a veterinary campus. To clarify the source of bacteria in the weasels, we examined the domestic animals reared in seven facilities on the campus. Extended-spectrum ß-lactamase (ESBL)-producing E. coli were isolated on deoxycholate hydrogen sulfide lactose agar, containing cephalexin (50 µg/mL) or cefotaxime (2 µg/mL), and were characterized with antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), replicon typing, and ß-lactamase typing analyses. Next-generation sequencing of the ESBL-encoding plasmids was also performed. CTX-M-14 producers isolated from both domestic animals and weasels were classified into six clusters with seven PFGE profiles. The PFGE and antimicrobial resistance profiles were characterized by the animal facility. All CTX-M-14 plasmids belonged to the IncI1 type with a similar size (98.9-99.3 kb), except for one plasmid that was 105.5 kb in length. The unweighted pair group method with arithmetic mean (UPGMA) revealed that the CTX-M-14 plasmid in the weasel isolates might have the same origin as the CTX-M-14 plasmid in the domestic animals. Our findings shed further light on the association of antimicrobial resistance between wild and domestic animals.

Two Novel Endornaviruses Co-infecting a Phytophthora Pathogen of Asparagus officinalis Modulate the Developmental Stages and Fungicide Sensitivities of the Host Oomycete.

Two novel endornaviruses, Phytophthora endornavirus 2 (PEV2) and Phytophthora endornavirus 3 (PEV3) were found in isolates of a Phytophthora pathogen of asparagus collected in Japan. A molecular phylogenetic analysis indicated that PEV2 and PEV3 belong to the genus Alphaendornavirus. The PEV2 and PEV3 genomes consist of 14,345 and 13,810 bp, and they contain single open reading frames of 4,640 and 4,603 codons, respectively. Their polyproteins contain the conserved domains of an RNA helicase, a UDP-glycosyltransferase, and an RNA-dependent RNA polymerase, which are conserved in other alphaendornaviruses. PEV2 is closely related to Brown algae endornavirus 2, whereas PEV3 is closely related to Phytophthora endornavirus 1 (PEV1), which infects a Phytophthora sp. specific to Douglas fir. PEV2 and PEV3 were detected at high titers in two original Phytophthora sp. isolates, and we found a sub-isolate with low titers of the viruses during subculture. We used the high- and low-titer isolates to evaluate the effects of the viruses on the growth, development, and fungicide sensitivities of the Phytophthora sp. host. The high-titer isolates produced smaller mycelial colonies and much higher numbers of zoosporangia than the low-titer isolate. These results suggest that PEV2 and PEV3 inhibited hyphal growth and stimulated zoosporangium formation. The high-titer isolates were more sensitive than the low-titer isolate to the fungicides benthiavalicarb-isopropyl, famoxadone, and chlorothalonil. In contrast, the high-titer isolates displayed lower sensitivity to the fungicide metalaxyl (an inhibitor of RNA polymerase I) when compared with the low-titer isolate. These results indicate that persistent infection with PEV2 and PEV3 may potentially affect the fungicide sensitivities of the host oomycete.

Metagenomic identification, sequencing, and genome analysis of porcine hepe-astroviruses (bastroviruses) in porcine feces in Japan.

Recently, hepe-astrovirus-like RNA viruses named bastroviruses (BastVs), have been found in human, pig, bat, and rat fecal samples. In this study, we determined nearly complete genome sequences of four BastVs in the feces of healthy pigs. Genetic characterization revealed that these porcine BastVs (PBastVs) and BastVs from other animals including humans, had the same genome organization, that is, they contained three predicted conserved domains of viral methyltransferase, RNA helicase, and RdRp in the nonstructural ORF1 and the astrovirus capsid domain in the structural ORF2. Phylogenetic analyses using RNA-dependent RNA polymerase and the capsid region revealed that PBastVs branched with bat and rat BastVs; however, the groups formed by each host were distantly related to human BastVs. Pairwise amino acid sequence comparison demonstrated that PBastVs shared 95.2-98.6% and 76.1-95.5% sequence identity among each other in the ORF1 and ORF2 regions, respectively; the sequence identities between PBastVs and BastVs from other animals were 21.4-42.5% and 9.1-20.6% in the ORF1 and ORF2 regions, respectively. This suggested that BastVs were derived from a common ancestor but evolved independently in each host population during a prolonged period. Putative recombination events were identified in the PBastV genome, suggesting that PBastVs gain sequence diversity and flexibility through recombination events. In an analysis of previously obtained metagenomic data, PBastV sequence reads were detected in 7.3% (23/315) of fecal samples from pigs indicating that PBastVs are distributed among pig populations in Japan.

Genetic diversity of enterovirus G detected in faecal samples of wild boars in Japan: identification of novel genotypes carrying a papain-like cysteine protease sequence.

The genetic diversity of enterovirus G (EV-G) was investigated in the wild-boar population in Japan. EV-G-specific reverse transcription PCR demonstrated 30 (37.5 %) positives out of 80 faecal samples. Of these, viral protein 1 (VP1) fragments of 20 samples were classified into G1 (3 samples), G4 (1 sample), G6 (2 samples), G8 (4 samples), G11 (1 sample), G12 (7 samples), G14 (1 sample) and G17 (1 sample), among which 11 samples had a papain-like cysteine protease (PL-CP) sequence, believed to be the first discoveries in G1 (2 samples) or G17 (1 sample) wild-boar EV-Gs, and in G8 (2 samples) or G12 (6 samples) EV-Gs from any animals. Sequences of the non-structural protein regions were similar among EV-Gs possessing the PL-CP sequence (PL-CP EV-Gs) regardless of genotype or origin, suggesting the existence of a common ancestor for these strains. Interestingly, for the two G8 and two G12 samples, the genome sequences contained two versions, with or without the PL-CP sequence, together with the homologous 2C/PL-CP and PL-CP/3A junction sequences, which may explain how the recombination and deletion of the PL-CP sequences occured in the PL-CP EV-G genomes. These findings shed light on the genetic plasticity and evolution of EV-G.

Mosquito-borne viruses, insect-specific flaviviruses (family Flaviviridae, genus Flavivirus), Banna virus (family Reoviridae, genus Seadornavirus), Bogor virus (unassigned member of family Permutotetraviridae), and alphamesoniviruses 2 and 3 (family Mesoniviridae, genus Alphamesonivirus) isolated from Indonesian mosquitoes.

Mosquitoes transmit many kinds of arboviruses (arthropod-borne viruses), and numerous arboviral diseases have become serious problems in Indonesia. In this study, we conducted surveillance of mosquito-borne viruses at several sites in Indonesia during 2016-2018 for risk assessment of arbovirus infection and analysis of virus biodiversity in mosquito populations. We collected 10,015 mosquitoes comprising at least 11 species from 4 genera. Major collected mosquito species were Culex quinquefasciatus, Aedes albopictus, Culex tritaeniorhynchus, Aedes aegypti, and Armigeres subalbatus. The collected mosquitoes were divided into 285 pools and used for virus isolation using two mammalian cell lines, Vero and BHK-21, and one mosquito cell line, C6/36. Seventy-two pools showed clear cytopathic effects only in C6/36 cells. Using RT-PCR and next-generation sequencing approaches, these isolates were identified as insect flaviviruses (family Flaviviridae, genus Flavivirus), Banna virus (family Reoviridae, genus Seadornavirus), new permutotetravirus (designed as Bogor virus) (family Permutotetraviridae, genus Alphapermutotetravirus), and alphamesoniviruses 2 and 3 (family Mesoniviridae, genus Alphamesonivirus). We believed that this large surveillance of mosquitoes and mosquito-borne viruses provides basic information for the prevention and control of emerging and re-emerging arboviral diseases.

Molecular characterization of full genome sequences of Newcastle disease viruses circulating among vaccinated chickens in Egypt during 2011-2013.

Although intensive vaccination programs have been implemented, Newcastle disease (ND) outbreaks, accompanied by severe economic losses, are still reported in Egypt. The genetic characterization of ND virus (NDV) strains isolated from ND-vaccinated chicken flocks provides essential information for improving ND control strategies. Therefore, here, 38 NDV strains were isolated and identified from outbreaks among vaccinated flocks of broiler chickens located in the provinces of Qena, Luxor, and Aswan of Upper Egypt during 2011-2013. The investigated broiler chicken flocks (aged 28 to 40 days) had high mortality rates of up to 80%. All NDV isolates were genetically analyzed using next-generation DNA sequencing. From these isolates, 10 representative NDV strains were selected for further genetic analyses. Phylogenetic analysis of full-length coding genes revealed that the Egyptian NDV isolates belonged to a single sub-genotype, VII.1.1. These isolates were phylogenetically distant from the vaccine strains, including La Sota or Clone 30 (genotype II), which have been commonly used to vaccinate chicken flocks. Amino acid substitution K78R was observed in the neutralizing epitopes of the F proteins; whereas several mutations were found in the neutralizing epitopes of the hemagglutinin-neuraminidase proteins, notably, E347K. Overall, our results suggested that the occurrence of neutralizing epitope variants may be one of potential reasons for ND outbreaks. Further studies are needed to determine the protective effect of current vaccines against circulating virulent NDV strains.

Comparative Analysis of Fecal Microbiota in Grasscutter (Thryonomys swinderianus) and Other Herbivorous Livestock in Ghana.

The grasscutter (also known as the greater cane rat; Thryonomys swinderianus) is a large rodent native to West Africa that is currently under domestication process for meat production. However, little is known about the physiology of this species. In the present study, aiming to provide information about gut microbiota of the grasscutter and better understand its physiology, we investigated the intestinal microbiota of grasscutters and compared it with that of other livestock (cattle, goat, rabbit, and sheep) using 16S rRNA metagenomics analysis. Similar to the other herbivorous animals, bacteria classified as Bacteroidales, Clostridiales, Ruminococcaceae, and Lachnospiraceae were abundant in the microbiome of grasscutters. However, Prevotella and Treponema bacteria, which have fiber fermentation ability, were especially abundant in grasscutters, where the relative abundance of these genera was higher than that in the other animals. The presence of these genera might confer grasscutters the ability to easily breakdown dietary fibers. Diets for grasscutters should be made from ingredients not consumed by humans to avoid competition for resources and the ability to digest fibers may allow the use of fiber-rich feed materials not used by humans. Our findings serve as reference and support future studies on changes in the gut microbiota of the grasscutter as domestication progresses in order to establish appropriate feeding methods and captivity conditions.

Comparative histological studies on properties of polysaccharides secreted by vomeronasal glands of eight Laurasiatheria species.

Most mammalian species have a vomeronasal organ that detects specific chemical substances, such as pheromones. Mucous fluid covering the vomeronasal sensory epithelium is secreted by vomeronasal glands, and the properties of these fluids have been suggested to be involved in chemical detection. Histological studies using periodic acid-Schiff (PAS) and Alcian blue pH 2.5 (AB) stains, which respectively detect natural and acidic polysaccharides, have suggested variations in the nature of the vomeronasal glands among species. Here, we investigated the responsivity of the vomeronasal glands to PAS and AB stains in eight Laurasiatheria species. All species studied herein possessed vomeronasal glands that stained positive for PAS, like other many reported species. The vomeronasal glands of dogs and minks - like rodents, were AB-negative, whereas those of cows, goats, sika deer, musk shrews and two bat species were positive. Considering the present findings and previous reports, the vomeronasal glands in most of Laurasiatheria species appear to be fundamentally abundant in acidic polysaccharides, whereas those in carnivores essentially contains neutral polysaccharides.

Bacterial and protozoan pathogens/symbionts in ticks infecting wild grasscutters (Thryonomys swinderianus) in Ghana.

Ticks and tick-borne pathogens constitute a great threat to livestock production and are a potential health hazard to humans. Grasscutters (Thryonomys swinderianus) are widely hunted for meat in Ghana and many other West and Central African countries. However, tick-borne zoonotic risks posed by wild grasscutters have not been assessed. The objective of this study was to investigate bacterial and protozoan pathogens in ticks infecting wild grasscutters. A total of 81 ticks were collected from three hunted grasscutters purchased from Kantamanto, the central bushmeat market in Accra. Ticks were identified as Ixodes aulacodi and Rhipicephalus sp. based on morphological keys, which were further confirmed by sequencing mitochondrial 16S ribosomal DNA (rDNA) and cytochrome oxidase I (COI) genes of specimens. Protozoan infections were tested by PCR amplifying 18S rDNA of Babesia/Theileria/Hepatozoon, while bacterial infections were evaluated by PCRs or real-time PCRs targeting Anaplasmataceae, Borrelia, spotted fever group rickettsiae, chlamydiae and Candidatus Midichloria mitochondrii. The results of PCR screening showed that 35.5% (27 out of 76) of I. aulacodi were positive for parasite infections. Sequencing analysis of the amplified products gave one identical sequence showing similarity with Babesia spp. reported from Africa. The Ca. M. mitochondrii endosymbiont was present in 85.5% (65 out of 76) of I. aulacodi but not in the five Rhipicephalus ticks. Two Anaplasmataceae bacteria genetically related to Ehrlichia muris and Anaplasma phagocytophilum were also detected in two I. aulacodi. None of the ticks were positive for Borrelia spp., spotted fever group rickettsiae and chlamydiae. Since I. aulacodi on wild grasscutters are potential carriers of tick-borne pathogens, some of which could be of zoonotic potential, rigorous tick control and pathogen analyses should be instituted especially when wild caught grasscutters are being used as foundation stock for breeding.