Characterization of a Tembusu Virus Isolated from Naturally Infected House Sparrows (Passer domesticus) in Northern China

The house sparrow (Passer domesticus) is one of the most widely distributed wild birds in China. Tembusu virus (TMUV) strain, TMUV‐SDHS, was isolated from house sparrows living around the poultry farms in Shandong Province, Northern China. Genetic analysis of E and NS5 genes showed that it had a close relationship with that of the YY5 strain, which can cause severe egg drop in ducks. Pathogenicity studies showed that the virus is highly virulent when experimentally inoculated into the ducks. These findings show that house sparrows carrying the Tembusu virus may play an important role in transmitting the virus among other species.     

Detection of New Delhi Metallo‐beta‐Lactamase and Extended‐Spectrum beta‐Lactamase Genes in Escherichia coli Isolated from Mastitic Milk Samples

In this study, eight Escherichia coli isolates were obtained from milk samples of dairy cattle suffering from clinical/subclinical mastitis. Isolates were characterized for antimicrobial resistance traits and virulence genes. Results revealed that one isolate was harbouring New Delhi metallo‐beta‐lactamase gene (bla_NDM). Cloning and sequencing of the PCR amplicon confirmed the identity of the gene (GenBank accession no. KC769583 ) having 100% homology with bla_NDM‐5 (GenBank accession no. JN104597.1 ), and this isolate was susceptible to colistin, chloramphenicol and tetracycline only. Moreover, another isolate carried extended‐spectrum beta‐lactamase (ESBL) gene – bla_CTX‐M, and all isolates possessed bla_TEM gene. Of the eight isolates, only one isolate was positive for shiga toxin gene (stx2), and none were harbouring stx1 gene. Occurrence of New Delhi metallo‐beta‐lactamase (bla_NDM) in one E. coli isolate and ESBL genes in other isolates poses a potential threat to human health following possible entry and spread through food chain.     

Molecular detection and characterization of carnivore parvoviruses in free‐ranging Eurasian otters (Lutra lutra) in southern Italy

The most important Italian population of the Eurasian otter (Lutra lutra) occurs in the southern part of the peninsula with two isolated sub‐populations of about 250 adult individuals. The Eurasian otter is considered to be near threatened and it is a fully protected species. The aims of this study were to investigate for the first time the occurrence and characterize the parvoviruses included in the species Carnivore protoparvovirus 1 in seven carcasses of road‐killed Eurasian otters from the southern Italy. Carnivore protoparvovirus 1 are responsible for acute gastroenteritis and leukopenia in pets and free‐ranging carnivores. Initial screening of tissue samples by real‐time PCR revealed CPV/FPV DNA in tissue samples of five Eurasian otters; three of them, showed co‐infections by both CPV and FPV. Among the five positive Eurasian otters, we successfully obtained six DNA sequences from four individuals including two CPV‐2a, one CPV‐2b, one CPV‐2c, and two FPV sequences. Comparison of these sequences with 250 VP2 gene sequences deposited in the GenBank database, showed 10 nt differences resulting in two synonymous and eight non‐synonymous substitutions. On the basis of these results, two sequences here found were characterized as new CPV‐2a, one was characterized as new CPV‐2b variant, and one was characterized as FPV‐like mutant. The last two sequences belong to a FPV and CPV‐2c strain respectively. Carnivore protoparvovirus 1 is reported for the first time in the Eurasian otter showing high infection value in southern Italy. Occurrence of this infection should be studied further to understand its possible pathogenicity and virulence to the fragile and isolate Eurasian otter population which live in southern Italy.     

Isolation and Genetic Characterization of a Tembusu Virus Strain Isolated From Mosquitoes in Shandong,China

Tembusu virus (TMUV) is a flavivirus, presumed to be a mosquito‐borne flavivirus of the Ntaya virus subgroup. To date, however, there have been no reports indicating that mosquitoes are involved in the spread of TMUV. In this study, we report the first isolation of TMUV from Culex mosquitoes. We describe the isolation and characterization of a field strain of TMUV from mosquitoes collected in Shandong Province, China. The virus isolate, named TMUV‐SDMS, grows well in mosquito cell line C6/36, in Vero and duck embryo fibroblast (DEF) cell lines, and causes significant cytopathic effects in these cell cultures. The TMUV‐SDMS genome is a single‐stranded RNA, 10 989 nt in length, consisting of a single open reading frame encoding a polyprotein of 3410 amino acids, with 5′ and 3′ untranslated regions of 142 and 617 nt, respectively. Phylogenetic analysis of the E and NS5 genes revealed that the TMUV‐SDMS is closely related to the TMUV YY5 and BYD strains which cause severe egg‐drop in ducks. The 3′NTR of TMUV‐SDMS contains two pairs of tandem repeat CS and one non‐duplicate CS, which have sequence similarities to the same repeats in the YY5 and BYD strains. Our findings indicate that mosquitoes carrying the TMUV may play an important role in the spread of this virus and in disease outbreak.     

Molecular Study of Dirofilaria immitis and Dirofilaria repens in Dogs from Tunisia

Dirofilaria immitis and Dirofilaria repens are mosquito‐borne nematodes which infect primarily dogs as their main definitive hosts. They cause cardiopulmonary (D. immitis) or cutaneous (D. repens) dirofilariasis in canids and other carnivores and can accidentally be transmitted to humans where they can induce a variety of clinical outcomes depending on organ localization. Dirofilaria spp. infection in dogs was assessed using molecular methods (PCR and sequencing) to identify the different Dirofilaria species occurring in 200 dogs from Northern and Central Tunisia. The overall molecular prevalence of Dirofilaria spp. was 17.5% (35/200). The prevalence of D. immitis (14.5%) was significantly higher than for D. repens (3%). Molecular prevalence of D. immitis was significantly higher in suburban compared to urban and rural regions. There was no difference in molecular prevalence of D. immitis or D. repens according to the dogs’ (sex or use). Dirofilaria immitis amplicons (accession numbers KR676386 ) fall into the same clade with D. immitis from China, India and Taiwan. Comparison of the partial sequences of D. repensITS2 rDNA gene ( KR676387 ) revealed 99.6% similarity with D. repens reported in dogs from USA. It had also 97.6% similarity with D. repens from mosquitoes in Czech Republic. High dog parasite burdens should motivate both medical doctors and veterinarians to consider these frequent infections.     

Genetic diversity of Bartonella spp. in vampire bats from Brazil

Recently, an increasing number of Bartonella species have been emerged to cause human diseases. Among animal reservoirs for Bartonella spp., bats stand out due to their high mobility, wide distribution, social behaviour and long‐life span. Although studies on the role of vampire bats in the epidemiology of rabies have been extensively investigated in Latin America, information on the circulation and genetic diversity of Bartonella species in these bat species is scarce. In the present work, 208 vampire bats, namely Desmodus rotundus (the common vampire bat; n = 167), Diphylla ecaudata (the hairy‐legged vampire bat; n = 32) and Diaemus youngii (the white‐winged vampire bat; n = 9) from 15 different states in Brazil were sampled. DNA was extracted from liver tissue samples and submitted to real‐time PCR (qPCR) and conventional PCR (cPCR) assays for Bartonella spp. targeting five genetic loci, followed by phylogenetic and genotype network analyses. Fifty‐one out of 208 liver samples (24.51%) were positive for Bartonella DNA in the ITS real‐time PCR assay [40 (78.43%) of them were from D. rotundus from 11 states, and 11 (21.57%) samples from D. ecaudata from three states. Eleven genotypes were found for each gltA and rpoB genes. Several ITS sequences detected in the present study clustered within the lineage that includes B. bacilliformis and B. ancachensis. The Bayesian phylogenetic inference based on the gltA gene positioned the obtained sequences in six different clades, closely related to Bartonella genotypes previously detected in D. rotundus and associated ectoparasites sampled in Latin America. On the other hand, the Bartonella rpoB genotypes clustered together with the ruminant species, B. schoenbuchensis and B. chomelii. The present study describes for the first time the molecular detection of Bartonella spp. in D. ecaudata bats. It also indicates that Bartonella spp. of vampire bats are genetically diverse and geographically widespread in Brazil.     

Uncommon Ehrlichia canis infection associated with morulae in neutrophils from naturally infected dogs in Brazil

Ehrlichia and Anaplasma species are the most common tick‐borne disease (TBD) pathogens in dogs worldwide. Ehrlichia canis, the aetiological agent of the Canine Monocytic Ehrlichiosis (CME), is known to replicate within the cytoplasm of mononuclear cells into clusters of organisms called morulae. However, detection of morulae in neutrophils is commonly observed in dogs infected by Ehrlichia ewingii or Anaplasma phagocytophilum. We report uncommon clinical cases of canine ehrlichiosis presenting morulae compatible with E. ewingii and A. phagocytophilum in dogs from two distinct regions of Brazil. Eight dogs were admitted to two veterinary teaching hospitals from Brazil, showing clinical or haematological signs suggestive of TBD. Blood or peritoneal fluid was withdrawn for haematological and cytologic analysis. All samples were evaluated by PCR assays for Ehrlichia and Anaplasma using genus‐specific primers for dsb, 16S rRNA and groEL genes, followed by sequencing. Samples were also evaluated by nested PCR assays for the 16S rRNA gene of E. ewingii and groEL gene of A. phagocytophilum and Anaplasma platys. Seven dogs revealed thrombocytopenia, six dogs had monocytosis and five presented lymphopenia and anaemia. All dogs showed morulae structures compatible with Ehrlichia spp. in neutrophils and were PCR‐positive for the dsb and 16S rRNA gene fragments of Ehrlichia, with sequences showing 100% identity with multiple E. canis sequences deposited in the GenBank™. Sequencing of 16S rRNA and groEL gene fragments from one PCR‐positive dog showed 100% identity with A. platys. Overall, our data suggest that in endemic regions for E. canis, that is Brazil, the presence of morulae in neutrophils may indicate infection by this bacterium. Herein, morulae were also found in neutrophils present in the peritoneal fluid of a dog. Also, this is the first report of E. canis and Hepatozoon canis co‐infection in neutrophils from naturally infected dogs confirmed by DNA sequencing.     

Rickettsia parkeri in free‐ranging wild canids from Brazilian Pampa

Spotted fevers are tick‐borne diseases associated with various Rickettsia species. Rickettsia parkeri sensu stricto (s.s.) is the agent of an emerging eschar‐associated rickettsiosis in humans from the USA and South American Pampa. Considering that R. parkeri s.s. is restricted to Americas and the potential role of dogs in the epidemiology of the disease, it is thus reasonable to hypothesize that wild canids could be involved in the enzootic cycle of this rickettsiosis. The aim of this work was to investigate the potential role of the wild canids from Pampa, Cerdocyon thous (crab‐eating fox) and Lycalopex gymnocercus (Pampas fox), in the ecology of R. parkeri s.s. For that, 32 live‐trapped free‐ranging wild canids were sampled. Ticks were observed in 30 of the 32 foxes. Of the 292 ticks collected, 22 (7.5%) were positive by PCR for the presence of R. parkeri s.s. DNA . Also, 20 (62%) wild canids showed antibodies against R. parkeri . The results suggest that wild canids are involved in the enzootic cycle of R. parkeri s.s. in the Pampa biome and could be responsible for pathogen (and its vectors) dispersal.     

Polymorphism at the ITS and NTS Loci of Perkinsus marinus Isolated from Cultivated Oyster Crassostrea corteziensis in Nayarit,Mexico and Phylogentic Relationship to P. marinus along the Atlantic Coast

Prevalence of the protozoan Perkinsus spp. in the gills of the pleasure oyster Crassostrea corteziensis from two estuaries in Nayarit, Mexico, was measured. The protozoan was identified by PCR amplification of the internal transcribed spacer (ITS) region of the rDNA of Perkinsus spp. The pathogen was found in 92% of oysters from Boca de Camichín and 77% of oysters from Pozo Chino. ITS sequences characterized from C. corteziensis showed 96–100% similarity to Perkinsus marinus. The most frequent ITS sequence (GenBank JQ266236 ) had 100% identity with the ITS locus of P. marinus from New Jersey, Maryland, South Carolina and Texas, and the second most frequent observed sequence (GenBank JQ266240 ) was 100% identical to ITS sequences of P. marinus from New Jersey, South Carolina, Louisiana, and Bahía Kino, Sonora, Mexico. The 14 sequences from the non‐transcribed spacer (NTS) showed 98% similarity to P. marinus from Texas. The most frequent polymorphism identified was at nucleotide 446 of the ITS region; however, the NTS showed the highest nucleotide diversity, thereby suggesting that this region is suitable for genotype identification. Moreover, the most conserved ITS marker is better for species‐specific diagnosis. Both the ITS and NTS sequences of P. marinus obtained from C. corteziensis were grouped in two clades, identifying two allelic variants of P. marinus.     

Antimicrobial Resistance of F4+ Escherichia Coli Isolated from Swine in Italy

Four‐hundred and forty‐two F4+ pathogenic Escherichia coli were isolated in a period of 10 years (2002–2011), from pigs that were suffering from diarrhoea belonging to Italian swine herds. The strains were analysed for their susceptibility to 12 antimicrobials using the disc diffusion method. During the study period, a statistically significant proportion of isolates resistant to enrofloxacin (14.5–89.3%), marbofloxacin (5.4–60.7%), flumequine (49.1–92.9%), danofloxacin (21.6–80%), florfenicol (9.8–64.3%), thiamphenicol (50–92%) and cefquinome (3.8–44%) was recorded. An increase in resistance (not statistically significant) to gentamicin (63.6–85.7%), apramycin (61.8–82.1%), trimethoprim‐sulphamethoxazole (75–89.3%), tetracycline (97–100%) and erythromycin (92.4–100%) was also observed. Based on antimicrobial multiresistance, the strains were collected into three groups: I. resistant to 2–5 antimicrobials; II. resistant to 6–8 antimicrobials; III. resistant to 9–12 antimicrobials. The number of isolates belonging to the first group showed a statistically significant decrease (P < 0.05; R² = 0.896; r = −0.9608), while the isolates belonging to the second and third groups showed a statistically significant increase in resistance (P < 0.05; R² = 0.753; r = 0.8890 and P < 0.05; R² = 0.727; r = 0.8701, respectively) over the period of study. The results of this study suggest the need for continued monitoring of the development of resistance.     

Intestinal parasites including Cryptosporidium,Cyclospora, Giardia,and Microsporidia,Entamoeba histolytica,Strongyloides, Schistosomiasis,and Echinococcus: Guidelines from the American Society of Transplantation Infectious Diseases Community of Practice

These updated guidelines from the Infectious Diseases Community of Practice of the American Society of Transplantation review the diagnosis, prevention, and management of intestinal parasites in the pre‐ and post‐transplant period. Intestinal parasites are prevalent in the developing regions of the world. With increasing travel to and from endemic regions, changing immigration patterns, and the expansion of transplant medicine in developing countries, they are increasingly recognized as a source of morbidity and mortality in solid‐organ transplant recipients. Parasitic infections may be acquired from the donor allograft, from reactivation, or from de novo acquisition post‐transplantation. Gastrointestinal multiplex assays have been developed; some of the panels include testing for Cryptosporidium, Cyclospora, Entamoeba histolytica, and Giardia, and the performance is comparable to conventional methods. A polymerase chain reaction test, not yet widely available, has also been developed to detect Strongyloides in stool samples. New recommendations have been developed to minimize the risk of Strongyloides donor‐derived events. Deceased donors with epidemiological risk factors should be screened for Strongyloides and recipients treated if positive as soon as the results are available. New therapeutic agents and studies addressing the optimal treatment regimen for solid‐organ transplant recipients are unmet needs.     

The Distribution of Escherichia coli Serovars,Virulence Genes,Gene Association and Combinations and Virulence Genes Encoding Serotypes in Pathogenic E. coli Recovered from Diarrhoeic Calves,Sheep and Goat

Ruminants, especially cattle, have been implicated as a principal reservoir of one of the enterovirulent Escherichia coli pathotypes. The detection of the virulence genes in diarrhoeic calves and small ruminants has not been studied in Egypt. To determine the occurrence, serotypes and the virulence gene markers, stx1, stx2, hylA, Flic_h7, stb, F41, K99, sta, F17, LT‐I, LT‐II and eae, rectal swabs were taken from diarrhoeic calves, sheep and goats and subjected to bacterial culture and PCR. The E. coli prevalence rate in the diarrhoeic animals was 63.6% in calves, 27.3% in goat and 9.1% in sheep. The 102 E. coli strains isolated from the calves, goat and sheep were 100% haemolytic non‐verotoxic and fitted into the Eagg group. The isolates belonged to seven O serogroups (O25, O78, O86, O119, O158, O164 and O157). The eae gene was detected in six of the strains isolated from the calves. The 102 bovine, ovine and caprine E. coli strains isolated in this study were negative for stx1, stx2, F41, LT‐I and Flic_h7 genes. The highest gene combinations were found to occur in the form of 24/102 isolates (23.5%) that carried the F17 gene predominantly associated with eaeA, hylA, K99 and Stb genes in the calves, while the hylA, K99 and Sta were the only genes found to be in conjunction in both calves and goats (6/102; 5.9% each). Our data show that in Egypt, large and small ruminants could be a potential source of infection in humans.     

Molecular epidemiology of Mycobacterium tuberculosis complex strains isolated from livestock and wild animals in Italy suggests the need for a different eradication strategy for bovine tuberculosis

Bovine tuberculosis (bTB ) is an important zoonosis, which has been re‐emerging in different ecological scenarios. In Sicily, Italy, from 2004 to 2014, an anatomopathological survey for tuberculosis‐like lesions both in farmed and wild animals was performed. The isolates were genotyped using spoligotyping and Mycobacterial Interspersed Repetitive Units‐Variable Number of Tandem Repeats (MIRU ‐VNTR ) techniques. High prevalence of lesions was observed for cattle (4%), pigs (4.9%) and wild boars (6.8%), and a total of 625 Mycobacterium bovis isolates were identified. Genotyping analysis showed the presence of 37 different spoligotypes including fifteen spoligotypes not present in other Italian regions and 266 MIRU ‐VNTR profiles. Spoligotype SB 0120 exhibited the highest prevalence in cattle (50%) and pigs (56%) and the highest genetic variety with 126 different MIRU ‐VNTR profiles. The isolation of M. bovis in a farmer underlines the importance of M. bovis identification during the human TB diagnostic processes. This study supported the use of the genotyping analysis as a valuable tool for the evaluation of the epidemiological role of pigs and other domestic reservoirs such as goats and the role of wildlife in the maintenance of bTB infection.     

First Detection of Antibodies Against African Swine Fever Virus in Faeces Samples

African swine fever (ASF) is a viral, highly lethal haemorrhagic disease of swine with no available vaccine or effective treatment. Introduction of ASF into a country triggers immediate restriction measures that cause significant economic losses and threatens spread to neighbouring countries. Wild boar populations have been recently assigned an essential role in the spread of African swine fever virus (ASFV) to European countries. Therefore, effective surveillance and monitoring of wild boar populations is required, but sampling wild boar is logistically challenging and expensive. This study assessed the feasibility of detecting antibodies against ASFV in faeces for later implementation in surveillance and control programmes. Two groups of pigs were experimentally infected with an attenuated ASFV isolate Ken05, and blood, oral fluid and faecal samples were tested for the presence of viral DNA using quantitative real‐time polymerase chain reaction (qPCR) to monitor infection progress. Faecal samples were analysed using two indirect enzyme‐linked immunosorbent assays (ELISAs) based on semipurified viral protein (vp) 72 or purified recombinant vp30 expressed in mammalian cells. Faecal samples from 9 of 10 pigs with non‐haemorrhagic diarrhoea tested positive for antibodies against ASFV using the two ELISA tests that showed a positive correlation. The serum sample results from the two indirect ELISAs were compared against results from the reference ELISA technique and the immunoperoxidase test. Our findings indicate the feasibility of faecal sampling for detecting anti‐ASFV antibodies, which may provide a practical non‐invasive alternative for sampling wild boar populations. In conclusion, the application of these ELISA tests to faecal field samples could be particularly useful to screen for the presence of ASF in field conditions.     

Tissue and blood protozoa including toxoplasmosis,Chagas disease,leishmaniasis, Babesia,Acanthamoeba, Balamuthia,and Naegleria in solid organ transplant recipients— Guidelines from the American Society of Transplantation Infectious Diseases Community of Practice

These updated guidelines from the Infectious Diseases Community of Practice of the American Society of Transplantation review the diagnosis, prevention, and management of tissue and blood protozoal infections in the pre‐ and post‐transplant period. Significant new developments in the field have made it necessary to divide the previous single guideline published in 2013 into two sections, with the intestinal parasites separated from this guideline devoted to tissue and blood protozoa. The current update reflects the increased focus on donor screening and risk‐based recipient monitoring for parasitic infections. Increased donor testing has led to new recommendations for recipient management of Toxoplasma gondii and Trypanosoma cruzi. Molecular diagnostics have impacted the field, with access to rapid diagnostic testing for malaria and polymerase chain reaction testing for Leishmania. Changes in Babesia treatment regimens in the immunocompromised host are outlined. The risk of donor transmission of free‐living amebae infection is reviewed. Changing immigration patterns and the expansion of transplant medicine in developing countries has contributed to the recognition of parasitic infections as an important threat to transplant outcomes. Medications such as benznidazole and miltefosine are now available to US prescribers as access to treatment of tissue and blood protozoa is increasingly prioritized.     

Pathogenic Rickettsia in ticks of spur‐thighed tortoise (Testudo graeca) sold in a Qatar live animal market

The dissemination of vector arthropods harbouring zoonotic pathogens through the uncontrolled transboundary trade of exotic and pet animals poses an important threat to Public Health. In the present report, we describe the introduction of pathogenic Rickettsia africae and R. aeschlimanni in ticks removed from imported tortoises in Qatar. A total of 21 ticks were collected from pet spur‐thighed tortoises (Testudo graeca) from Doha, May 2018, and studied for species identification and characterization of Rickettsia spp. Morphological and molecular analysis of ticks allowed their identification as Hyalomma aegyptium. Molecular analysis of partial ompA and gltA genes showed that Rickettsia sequences found on these ticks clustered with sequences classified as R. aeschilimanii and R. africae. Since pre‐adult stages of H. aegyptium also feed on humans, this tick species may play a role in the transmission of R. aeschilimanii and R. africae. We alert for the introduction of non‐native pets as vehicles for tick importation, known vectors for animal and human pathogenic agents. Importation of exotic species into non‐autochthonous countries deserves strict control to enforce robust surveillance and mitigate potential exotic diseases epidemics.     

Genomic epidemiology of the emerging pathogen Batrachochytrium dendrobatidis from native and invasive amphibian species in Chile

Emerging fungal diseases represent a threat to food security, animal and human health worldwide. Amphibian chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd ), has been associated with catastrophic and well‐documented amphibian population declines and extinctions. For the first time, Bd was cultured from native and non‐native wild amphibians in Chile. Phylogenomic analyses revealed that Chilean isolates AVS2, AVS4 and AVS7 group within the global panzootic lineage of Bd (Bd GPL) in a single highly supported clade that includes a genotype previously isolated from the United Kingdom. Our results extend the known distribution of Bd GPL in South America and suggest a single and relatively recent introduction of Bd GPL into the country, providing additional support to the role of anthropogenic activity in the global spread of this panzootic lineage.