The information on microbiota composition in the human gastrointestinal tract predominantly originates from the analyses of human faeces by application of next generation sequencing (NGS). However, the detected composition of the faecal bacterial community can be affected by various factors including experimental design and procedures. This study evaluated the performance of different protocols for collection and storage of faecal samples (native and OMNIgene.GUT system) and bacterial DNA extraction (MP Biomedicals, QIAGEN and MO BIO kits), using two NGS platforms for 16S rRNA gene sequencing (Ilumina MiSeq and Ion Torrent PGM). OMNIgene.GUT proved as a reliable and convenient system for collection and storage of faecal samples although favouring Sutterella genus. MP provided superior DNA yield and quality, MO BIO depleted Gram positive organisms while using QIAGEN with OMNIgene.GUT resulted in greatest variability compared to other two kits. MiSeq and IT platforms in their supplier recommended setups provided comparable reproducibility of donor faecal microbiota. The differences included higher diversity observed with MiSeq and increased capacity of MiSeq to detect Akkermansia muciniphila, [Odoribacteraceae], Erysipelotrichaceae and Ruminococcaceae (primarily Faecalibacterium prausnitzii). The results of our study could assist the investigators using NGS technologies to make informed decisions on appropriate tools for their experimental pipelines.

Lumpy skin disease caused by Capripoxvirus is at the moment the most important threat to European cattle industry. The only way for successful control of disease is fast and efficient diagnosis and vaccination. According to EU legislation, vaccination against LDS can be conducted only after confirmation of the disease. Croatia has a special position regarding LSD-in 2016, for the first-time vaccination of the entire cattle population was conducted without an index case. The presence of vaccine viral particles was detected in milk, skin nodules, blood and nasal swabs in seven from total of eight herds. The presence of virus genome was detected in five cows from 10 up to 21-day post-vaccination. The virus was successfully isolated on cell culture from 10 up to 21-day post-vaccination from three animals. The obtained results support the need for further efforts to develop safer vaccines against LSDV.

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Due to SARS CoV-2 recombination rates, number of infected people and recent reports of environmental contamination, the possibility of SARS CoV-2 transmission to animals can be expected. We tested samples of dominant free-living and captive wildlife species in Croatia for the presence of anti-SARS CoV-2 antibodies and viral RNA. In total, from June 2020 until February 2021, we tested blood, muscle extract and fecal samples of 422 free-living wild boars (Sus scrofa), red foxes (Vulpes vulpes) and jackals (Canis aureus); blood and cloacal swabs of 111 yellow-legged gulls (Larus michahellis) and fecal samples of 32 zoo animals. A commercially available ELISA (ID.Vet, France) and as a confirmatory test, a surrogate virus neutralization test (sVNT; GenScript, Netherlands) were used. Fecal samples were tested for the presence of viral RNA by a real-time RT–PCR protocol. Fifteen out of 533 (2.8%) positive ELISA results were detected; in wild boars (3.9%), red foxes (2.9%) and jackals (4.6%). However, the positive findings were not confirmed by sVNT. No viral RNA was found. In conclusion, no spillover occurred within the investigated period (second COVID-19 wave). However, further investigation is needed, especially regarding wildlife sample features for serological tests.

The first detection of avian nephritis virus (ANV) in goose embryos and of turkey astrovirus-1 (TAstV-1) in duck embryos is described. Intestinal samples from duck and goose embryos from five duck and four goose flocks in Croatia were tested by polymerase chain reaction for the presence of ANV, TAstV-1, turkey astrovirus-2, chicken astrovirus, duck astrovirus and also for the presence of avian reovirus, Derzsy's disease virus and duck enteritis virus. The kidneys from duck and goose embryos were also tested for ANV, while liver samples were tested for duck astrovirus. Duck embryos were also tested to detect duck circovirus and goose embryos for the presence of goose circovirus and goose haemorrhagic polyomavirus. All embryos were in the final stage of incubation and were characterized by moderate to markedly retarded growth. ANV was confirmed in the intestines and kidneys of embryos from two duck and two goose flocks and TAstV-1 was found in embryos from two duck flocks. One duck flock was positive for both ANV and TAstV-1. No other viruses were found in tested flocks. Phylogenetic analysis based on the ANV polymerase gene fragment of ANV sequences detected in duck and goose embryos revealed greatest similarity (88.1 to 97.2%) with ANV isolates from chickens. Further, the existence of at least two types of ANV circulating in Croatian duck and goose flocks was confirmed. Based on the phylogenetic analysis of the portion of TAstV-1 polymerase gene, two detected TAstV-1 nucleotide sequences were 99.5% similar. Compared with six TAstV-1 sequences, Croatian sequences showed one unique nucleotide change. In addition to other possible causes of stunted growth and late embryonic death, these findings suggest that ANV and/or TAstV-1 infection may be a contributing factor in the pre-hatching mortality of ducklings and goslings.