Emergence of Group B Streptococcus Disease in Pigs and Porcupines, Italy.

We describe group B Streptococcus linked to disease in farmed pigs and wild porcupines in Italy. Occurrence in pigs was attributed to transmission from nonpasteurized bovine milk whey. Antimicrobial-resistance profiles in isolates from porcupines suggest no common source of infection. Our findings expand the known host range for group B Streptococcus disease.

Preliminary investigation on enzymatic activity in saliva of Hystrix cristata L., 1758.

Mammal's saliva contains a variety of electrolytes and proteins. They carry out an important role in the digestion process, in the antibacterial and antiviral activity, in lubrication and maintenance of oral general health status. It may also contain several enzymes according to dietary habits and general wellness. Sialochemistry is a valid alternative to the haematochemical analysis for the evaluation of animal health and nutritional status. At present, very little knowledge is available on health status and pathology of crested porcupine (Hystrix cristata) and no data are yet available on salivary enzymes. Between 2018 and 2020, a preliminary investigation of enzymatic activity on saliva samples was carried out from captured porcupines. In crested porcupine saliva, enzymatic activity of trypsin, chymotrypsin, N-Aminopeptidase, amylase, lignin peroxidise, cellulase and chitinase were recorded. Superoxide dismutase, catalase, glutathione S-transferase and alkaline phosphatase activity was also detected. The superoxide dismutase activity resulted higher (3.13 SD 3.58 U/mg proteins) than those of catalase (130.80 SD 110.65 mU/mg proteins) and glutathione S-transferase (20.21 SD 16.62 mM/mg proteins). Alkaline phosphatase activity resulted lower (5.91 SD 6.12 mU/mg proteins) than acidic phosphatase (19.00 SD 16.16 U/mg proteins) with the highest values of saliva alkaline phosphatases recorded in young individuals. These preliminary data bring new knowledge on crested porcupine saliva enzymes and may provide a useful tool for further investigation on the adaptive response of crested porcupine to different environmental condition and diet. Additional investigation concerning a possible alternative use of saliva enzymes as indicator of health and nutritional status of this rodent are desirable.

Prevalence, genetic diversity and implications for public health of Enterocytozoon bieneusi in various rodents from Hainan Province, China.

BACKGROUND: Rodents, globally overpopulated, are an important source for zoonotic disease transmission to humans, including Enterocytozoon bieneusi (one of the most prevalent zoonotic pathogens). Here, we studied the prevalence and performed genetic analyses of E. bieneusi in rodents from the Hainan Province of China. METHODS: A total of 603 fresh fecal samples were gathered from 369 wild rats, 117 bamboo rats, 93 Asiatic brush-tailed porcupine and 24 red-bellied squirrels. The wild rats were identified to the species level by amplification of a 421-bp region of the cytb gene from fecal DNA using PCR. Genotype analysis was performed by amplification of the internal transcribed spacer (ITS) region of rDNA of E. bieneusi using PCR. RESULTS: Seven wild rat species were identified. The average rate of infection with E. bieneusi was 15.8% (95/603) with 18.7% (69/369) in wild rats, 11.9% (25/210) in farmed rodents and 4.2% (1/24) in red-bellied squirrels. Sixteen E. bieneusi genotypes were identified, including 9 known genotypes (D, Type IV, PigEBITS7, Peru8, Peru11, ESH02, S7, EbpA and CHG5), and 7 novel genotypes (HNR-I to HNR-VII). Genotype D (44.2%, 42/95) predominated, followed by PigEBITS7 (20.0%, 19/95), HNR-VII (15.8%, 15/95), Type IV (5.3%, 5/95), HNR-III (2.1%, 2/95), HNR-VI (2.1%, 2/95) and each of the remaining 10 genotypes (1.1%, 1/95). The phylogenetic analysis of the ITS region of E. bieneusi divided the identified genotypes into the following four groups: Group 1 (n = 13), Group 2 (n = 1), Group 12 (n = 1), and the novel Group 13 (n = 1). CONCLUSIONS: To our knowledge, this is the first report on the identification of E. bieneusi in rodents from Hainan, China. The zoonotic potential of the identified E. bieneusi genotypes suggested that the rodents poses a serious threat to the local inhabitants. Thus, measures need to be taken to control the population of wild rats in the areas investigated in this study, along with identification of safe methods for disposal of farmed rodent feces. Additionally, the local people should be made aware of the risk of disease transmission from rodents to humans.

Crested Porcupine (Hystrix cristata L.): A New Potential Host for Pathogenic Leptospira Among Semi-Fossorial Mammals.

Wildlife plays a pivot role in the epidemiology of leptospirosis and rodents have a reservoir function for several Leptospira serogroups. The crested porcupine is the largest rodent of the Italian fauna and shares the same environment with red foxes, badgers, coypus and wild boars that are known to be infected by Leptospira. Between 2018 and 2019 the seroprevalence of Leptospira in crested porcupine was investigated for the first time. Overall 7 out of 14 sera (50 %) were found positive to Leptospira. Icterohaemorrhagiae resulted as the most detected serogroup (57 %) followed by Pomona, Australis and Sejroe. The highest titer (1:1600) was detected for the serogroup Australis. These results indicate that porcupines could be infected by several serogroup of Leptospira and the role of reservoir or accidental host need to be addressed. Further investigations are necessary in order to clarify the leptospirosis - epidemiology - wildlife framework in light of its potential zoonotic source.

Detection of Tetracycline Resistance Genes in European Hedgehogs (Erinaceus europaeus) and Crested Porcupines (Hystrix cristata).

Relatively little is known regarding the role of wildlife in the development of antibiotic resistance. Our aim was to assess the presence of the tetracycline resistance genes, tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(K), tet(L), tet(M), tet(O), tet(P), tet(Q), tet(S), and tet(X), in tissue samples of 14 hedgehogs (Erinaceus europaeus) and 15 crested porcupines (Hystrix cristata) using PCR assays. One or more tet genes were found in all but three hedgehogs and one crested porcupine. Of the 14 tetracycline resistance genes investigated, 13 were found in at least one sample; tet(G) was not detected. We confirmed the potential role of wild animals as bioindicators, reservoirs, or vectors of antibiotic-resistant bacteria in the environment.

Characterization of novel lignocellulose-degrading enzymes from the porcupine microbiome using synthetic metagenomics.

Plant cell walls are composed of cellulose, hemicellulose, and lignin, collectively known as lignocellulose. Microorganisms degrade lignocellulose to liberate sugars to meet metabolic demands. Using a metagenomic sequencing approach, we previously demonstrated that the microbiome of the North American porcupine (Erethizon dorsatum) is replete with genes that could encode lignocellulose-degrading enzymes. Here, we report the identification, synthesis and partial characterization of four novel genes from the porcupine microbiome encoding putative lignocellulose-degrading enzymes: ß-glucosidase, α-L-arabinofuranosidase, ß-xylosidase, and endo-1,4-ß-xylanase. These genes were identified via conserved catalytic domains associated with cellulose- and hemicellulose-degradation. Phylogenetic trees were created for each of these putative enzymes to depict genetic relatedness to known enzymes. Candidate genes were synthesized and cloned into plasmid expression vectors for inducible protein expression and secretion. The putative ß-glucosidase fusion protein was efficiently secreted but did not permit Escherichia coli (E. coli) to use cellobiose as a sole carbon source, nor did the affinity purified enzyme cleave p-Nitrophenyl ß-D-glucopyranoside (p-NPG) substrate in vitro over a range of physiological pH levels (pH 5-7). The putative hemicellulose-degrading ß-xylosidase and α-L-arabinofuranosidase enzymes also lacked in vitro enzyme activity, but the affinity purified endo-1,4-ß-xylanase protein cleaved a 6-chloro-4-methylumbelliferyl xylobioside substrate in acidic and neutral conditions, with maximal activity at pH 7. At this optimal pH, KM, Vmax, and kcat were determined to be 32.005 ± 4.72 µM, 1.16x10-5 ± 3.55x10-7 M/s, and 94.72 s-1, respectively. Thus, our pipeline enabled successful identification and characterization of a novel hemicellulose-degrading enzyme from the porcupine microbiome. Progress towards the goal of introducing a complete lignocellulose-degradation pathway into E. coli will be accelerated by combining synthetic metagenomic approaches with functional metagenomic library screening, which can identify novel enzymes unrelated to those found in available databases.

The dorsal skin structure contributes to the surface bacteria populations of Sunda Porcupine (Hystrix javanica).

Skin becomes the largest organ in the body and protects its own inner layer. The structure and chemical composition of the skin contribute to skin condition and affect the habitat of certain bacteria. The Sunda Porcupine is one of endemic animals of Indonesia which possesses quill as the main derivate of its skin and as a defence tool against predators. The present study used nine adults (five females and four males) of Sunda Porcupine and aimed to observe the correlation of skin structure with bacterial population at the surface level. The skin was wavy due to the protrusion of quill follicle orifices on the skin surface and formed clusters. The skin of Sunda Porcupine was also wrinkled and had a lot of flakiness. Histologically, the skin was composed of epidermis, dermis, hypodermis and subcutaneous muscle. The quill follicles and their properties were the dominant structure component of the skin. No sweat gland was observed in the skin of the Sunda Porcupine, and sebaceous gland was found only around quill and hair follicles. The bacterias identified in the skin were Staphylococcus aureus, S. epidermidis, Micrococcus sp. and Salmonella sp. When compared, the bacterial population was higher in the lumbosacral region than in the thoracodorsal region, but the difference was not significant. The density of quill clusters was negatively correlated to the bacterial population. It was suggested the structure of the skin has contribution to bacterial population in dorsal trunk of the Sunda Porcupine.

Crested porcupines (Hystrix cristata): mycophagist spore dispersers of the ectomycorrhizal truffle Tuber aestivum.

Truffles, as hypogeous, ectomycorrhizal fungi, have no means to actively discharge spores into the environment and thus depend on mycophagists for spore dispersal. After consumption of fruiting bodies by animals and passage through the digestive tract, the spores are released in faecal pellets. Recently, in the Abruzzo region (Italy), Hystrix cristata has been spotted inside private truffières, but its role in spore dispersal has never been investigated. Here, we report our research on the occurrence of Tuber aestivum spores in porcupine's faecal contents in a truffière in L'Aquila, Italy, where a H. cristata specimen was photographed. The spores were isolated from faeces by using a suspension of 0.7 M ZnSO4. We also verified degradation and disfiguration of the digested spores' reticular ornamentation compared to that of fresh spores from ascomata collected inside the truffière, through measurements performed by scanning electron microscopy. A few truffle spores had germinated within the faeces.

Micrococcoides hystricis gen. nov., sp. nov., a novel member of the family Micrococcaceae, phylum Actinobacteria.

A Gram-stain-positive bacterium, designated TSL3T, was isolated from faeces of a porcupine, Hystrix indica, from the Budapest Zoo and Botanical Garden, Hungary. On the basis of 16S rRNA gene sequence analysis, the strain is phylogenetically related to the family Micrococcaceae. The highest 16S rRNA gene sequence similarity was found with Micrococcus terreus V3M1T (96.50 %) followed by Arthrobacter humicola KV-653T (96.43 %). Cells of strain TSL3T were aerobic, non-motile and coccoid-shaped. The main fatty acids were anteiso-C15 : 0 (54.4 %), iso-C16 : 0 (18.2 %) and iso C15 : 0 (9.7 %). The major menaquinone was MK-7, and the polar lipid profile included phosphatidylglycerol, diphosphatidylglycerol, dimannosylglyceride, trimannosyldiacylglycerol, phosphatidylinositol, three unknown phospholipids and two unknown glycolipids. Strain TSL3T showed the peptidoglycan structure A4alpha l-Lys - Gly - l-Glu. The DNA G+C content of strain TSL3T was 58.4 mol%. Phenotypic and genotypic characterisation clearly showed that strain TSL3T could be differerentiated from the members of other genera in the family Micrococcaceae. According to these results, strain TSL3T represents a novel genus and species, for which the name Micrococcoides hystricis gen. nov., sp. nov. is proposed. The type strain is TSL3T (=DSM 29785T=NCAIM B. 02604T).

Short Communication: Elucidation of bacterial community structure on thin-spined porcupine (Chaetomys subspinosus) spines by denaturing.

Thin-spined porcupines (Chaetomys subspinosus) are threatened with extinction and are categorized as vulnerable. This is because of alteration to and loss of their habitat and possible hunting activities in their distribution area. Their spines constitute one of their defense mechanisms, which can be fomites for pathogens to humans. However, little is known about such pathogens. The present study aimed to detect bacteria on spines of C. subspinosus, from the Una Biological Reserve, South of Bahia, northeastern Brazil, by analyzing metagenomic DNA, isolating bacterial culture, using the denaturing gradient gel electrophoresis (DGGE) technique, and sequencing. Six anatomical points were selected for withdrawing spine samples from an individual C. subspinosus. At all sample points, bacteria were detected by bacteriological culture and/or DGGE and sequencing of excised bands. When all samples were combined, standard PCR-DGGE analysis of bacteria present in the spines identified 15 distinct bands, thereby revealing a distinct bacterial community. The main pathogens identified through sequencing were Bacillus cereus, B. thuringiensis, B. anthracis, and B. pumilus. The present study demonstrated the isolation and identification of non-pathogenic and pathogenic bacteria on the spines of C. subspinosus.

Azole-resistant Candida albicans from a wild Brazilian porcupine (Coendou prehensilis): a sign of an environmental imbalance?

This study aimed at evaluating the in vitro antifungal susceptibility of Candida albicans isolates obtained during necropsy of a wild Brazilian porcupine and the mechanism of azole resistance. Initially, we investigated the in vitro susceptibility of the three isolates to amphotericin B, caspofungin, fluconazole, itraconazole, ketoconazole and voriconazole. Afterwards, three sub-inhibitory concentrations (47, 21 and 12 mg/l) of promethazine, an efflux pump inhibitor, were tested in combination with the antifungal drugs in order to evaluate the role of these pumps in the development of antifungal resistance. In addition, the three isolates were submitted to RAPD-PCR and M13-fingerprinting analyses. The minimum inhibitory concentrations (MICs) obtained with the isolates were 1, 0.03125, 250, 125, 8 and 250 mg/l for amphotericin B, caspofungin, fluconazole, itraconazole, ketoconazole and voriconazole, respectively, and the isolates were found to be resistant to all tested azoles. The addition of the three subinhibitory concentrations of promethazine resulted in statistically significant (P < 0.05) reductions in the MICs for all tested drugs, with decreases to azoles being statistically greater than those for amphotericin B and caspofungin (P < 0.05). The molecular analyses showed a genetic similarity among the three tested isolates, suggesting the occurrence of candidemia in the studied animal. These findings highlight the importance of monitoring antifungal susceptibility of Candida spp. from veterinary sources, especially as they may indicate the occurrence of primary azole resistance even in wild animals.

Disseminated pulmonary adiaspiromycosis in a crested porcupine (Hystrix cristata Linnaeus, 1758).

Adiaspiromycosis is primarily a necrotizing granulomatous pneumonia caused by a dimorphic fungus of the genus Emmonsia. A young crested porcupine (Hystrix cristata) found dead showed multiple fractures, chronic pleuritis, and granulomatous pneumonia. Microscopically, cystic structures were consistent with adiaspiromycosis by Emmonsia crescens. The diagnosis was confirmed using molecular methods.

An intrafamilial transmission of Arthroderma benhamiae in Canadian porcupines (Erethizon dorsatum) in a Japanese zoo.

An intra-familial transmission of Arthroderma benhamiae in Canadian porcupines (Erethizon dorsatum) housed in a Japanese zoo was studied. The family consisted of an adult couple and two offspring (a male and a female). The porcupettes, born in Japan, showed severe hair loss while the parent animals, imported from the USA. (male) and Canada (female), showed mild symptoms or were asymptomatic. Morphologically identical Tricophyton spp. isolates were recovered within seven days from quills of all animals on chloramphenicol-supplemented potato dextrose agar plates incubated at 37 degrees C. Two representative colonies from each animal were identified as Arthroderma benhamiae Americano-European race based on mating type (+) and the internal transcribed spacer (ITS) 1-5.5S-ITS 2 region of the rRNA gene sequences (AB236404-AB236408). The present cases constituted the second isolation of dermatophytes from porcupines. There were two different ITS types, i.e., the predominant one isolated from all animals and a secondary one recovered from only the mother porcupine. The sequences have never been recorded in Japan or in the GenBank database to the best of our knowledge. In addition, they were located at a cluster involving the type strain and mating strains of A. benhamiae Americano-European race and its F1 progeny. In contrast, 28 rodents (eight species) and three insectivora (1 species) exhibited in the petting zoo were negative for any dermatophytes as determined by culture.

Toxoplasma gondii in an African crested porcupine (Hystrix cristata).

An adult female crested porcupine (Hystrix cristata) was evaluated for acute onset of neurologic signs including head tilt, circling, and ataxia. She was found dead in her holding area 2 days after initially exhibiting clinical signs. Necropsy was unremarkable. Histopathology of brain tissue revealed the presence of protozoal cysts associated with inflammation as the underlying cause of clinical signs and death. Immunohistochemical staining of brain tissue for Toxoplasma gondii was strongly positive. PCR on fresh brain confirmed T. gondii as the causative organism. An adult male in the same enclosure has demonstrated similar neurologic signs over the past 3 years and has failed to respond to various medical treatments. Clinical disease associated with T. gondii has not been previously reported in this porcupine species or any other Old World porcupines, although there are several reports of clinical toxoplasmosis involving New World porcupine species.