Abstract
Viral enteric pathogens continuously burden intensive pig farming, causing gastrointestinal diseases of epidemic and endemic nature. The present study investigated two diarrhoea outbreaks on ...a large farrow-to-finish holding and subsequent circulation of outbreak-related enteric viruses. These viruses were characterised by whole genome sequencing, and statistical evaluation of the impact on specific production metrics was performed. The results provided evidence that the
Porcine epidemic diarrhoea virus
–swine enteric coronavirus (PEDV–SeCoV) S gene recombinant strain was responsible for the first outbreak, whilst
Rotavirus A
(RVA) in a mixed infection with
Rotavirus B
(RVB) and porcine kobuvirus (PKV) probably caused the second diarrhoea outbreak. Whole genome characterisation revealed a porcine origin of all viruses involved and significant heterogeneity of RVB strain, proposing four novel genotypes and changes in RVB VP1 genotype classification. The statistical evaluation confirmed only a minor disturbance in the number of weaned pigs per sow, with statistical forecasting showing positive trends. A follow-up study corroborated the endemicity of RVA and PKV, in contrast to PEDV–SeCoV. Punctual, comprehensive and timely investigation of diarrhoea outbreaks is a prerequisite for applying adequate pig health and biosecurity management. Calculating such outbreaks' impact on production metrics can potentially shape future decisions on management improvements.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
To fulfill epidemiological data and investigate possible interspecies transmission, this study shall attempt to sequence representative HEV strains of human, swine and wild boar origin collected from ...2010 to 2017 in Croatia.
In total, 174 anti-HEV antibody positive human sera samples; 1419 blood or faeces samples of swine, as well as 720 tissue and/or blood samples of wild boar originating from different counties (18 in total) in Croatia were tested for the presence of HEV RNA.
HEV RNA was detected in 26 human sera samples (14.9%; 95% CI 10.4-21.0%). HEV RNA was detected in 216 tested swine (15.2%; 95% CI 13.5-17.1%), regardless of age, farm breeding system or geographical origin. Viral RNA was also detectable in faeces samples which prove that swine actively participate in shedding HEV into the environment. Of the total of 720 tested wild boar samples, 83 were HEV RNA positive (11.5, 95% CI 9.4-14.1%) originating from six counties. According to the sequence analysis all strains have shown to be members of Orthohepevirus A genotype HEV-3, regardless of host. The genotyping results confirm grouping of sequences into four subtypes of HEV strains of which subtypes 3a and 3c belong to the general cluster 3abchij, and were predominately detected during the study, while subtypes 3e and 3f fall within cluster 3efg. Strains within subtypes 3a and 3e were found in humans, swine and wild boars; subtype 3c strains were derived from humans and swine, whereas subtype 3f strains were found only in humans. Strains belonging to subtypes 3a and 3c were derived during the entire investigated period and may be considered endemic in Croatia, whereas strains within subtypes 3e and 3f were detected sporadically indicating the possibility of newly imported infections.
All detected strains show to be genetically highly related to strains found in humans and/or animals from other European Countries, indicating that trade of live animals or wild boar movement increases the risk of HEV infection spread. Furthermore, homologous strains found in different investigated species within this study indicate interspecies transmission of HEV and/or an existence of an accessible mutual source of infection.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Common carp (Cyprinus carpio) is a very important fish species for warm‐water aquaculture in Croatia. All Croatian carp farms are subjected to a surveillance programme for the presence of koi ...herpesvirus (KHV), causing a deadly disease called koi herpesvirus disease (KHVD). However, there is no surveillance for other viral pathogens of importance like carp edema virus (CEV), a causative agent of koi sleepy disease (KSD). During regular testing within the KHVD surveillance programme, we tested samples for CEV simultaneously. The screening indicated possible outbreaks of KHVD and KSD. During 2016, KHVD broke out in an isolated area and soon thereafter a KHV eradication programme was successfully performed. However, during 2018 and 2019, two additional mortality events occurred in lakes in the southern part of Croatia during the spring. Samples from both events tested positive for CEV. An epidemiological investigation confirmed the introduction of infected carps from an infected farm to one of the lakes. To prevent the spreading of CEV into open waters, it is of utmost importance to introduce CEV testing before fish movement or to perform regular testing of all carp farms in the country to determine CEV prevalence for the purpose of implementation of control measures.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The influence of cultivation on the expression pattern of canine adipose-derived mesenchymal stem cells (cAD-MSCs) surface markers, contributing to, among others, the promotion of growth, ...proliferation, differentiation and immunomodulatory mechanisms of an excellent therapeutic, is still unknown. To fill the gap, we investigated CD90, CD44, CD73, CD29, CD271, CD105, CD45 and CD14 patterns of expression at the protein level with flow cytometry and mRNA level using a real-time polymerase chain reaction array. Gentle variations of expression occurred during cultivation, along with increased CD90, CD44 and CD29 expression, low and decreasing CD271 and CD73 expression and a decrease of initially high CD105. As expected, CD45 and CD14 were not expressed by cAD-MSCs. Interestingly, we discovered a significant decrease of CD73 expression, compared to early (P1–P3) to late (P4–P6) passages, although the CD73 gene expression was found to be stable. The percentage of positive cells was found to be higher for all positive markers up to P4. As CD73′s one important feature is a modulation from a pro-inflammatory environment to an anti-inflammatory milieu, the expression of CD73 in our conditions indicate the need to consider the time cells spend in vitro before being transplanted into patients, since it could impact their favourable therapeutical properties.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
As a leading viral cause of acute gastroenteritis in both humans and pigs, rotavirus A (RVA) poses a potential public health concern. Although zoonotic spillover of porcine RVA strains to humans is ...sporadic, it has been detected worldwide. The origin of chimeric human-animal strains of RVA is closely linked to the crucial role of mixed genotypes in driving reassortment and homologous recombination, which play a major role in shaping the genetic diversity of RVA. To better understand how genetically intertwined porcine and zoonotic human-derived G4P6 RVA strains are, the present study employed a spatiotemporal approach to whole-genome characterization of RVA strains collected during three consecutive RVA seasons in Croatia (2018-2021). Notably, sampled children under 2 years of age and weanling piglets with diarrhea were included in the study. In addition to samples tested by real-time RT-PCR, genotyping of VP7 and VP4 gene segments was conducted. The unusual genotype combinations detected in the initial screening, including three human and three porcine G4P6 strains, were subjected to next-generation sequencing, followed by phylogenetic analysis of all gene segments, and intragenic recombination analysis. Results showed a porcine or porcine-like origin for each of the eleven gene segments in all six RVA strains. The G4P6 RVA strains detected in children most likely resulted from porcine-to-human interspecies transmission. Furthermore, the genetic diversity of Croatian porcine and porcine-like human G4P6 strains was propelled by reassortment events between porcine and porcine-like human G4P6 RVA strains, along with homologous intragenotype and intergenotype recombinations in VP4, NSP1, and NSP3 segments. Described concurrent spatiotemporal approach in investigating autochthonous human and animal RVA strains is essential in drawing relevant conclusions about their phylogeographical relationship. Therefore, continuous surveillance of RVA, following the One Health principles, may provide relevant data for assessing the impact on the protectiveness of currently available vaccines.
Rotavirus A (RVA) is an important pathogen for porcine health. In comparison to humans, RVA in domestic animals and especially in wildlife is under researched. Therefore, the aim of the present study ...was to investigate the prevalence, genetic diversity, molecular epidemiology and interspecies transmission of RVA in domestic pigs and wild boars. During the three consecutive RVA seasons (2018–2021) we collected 445 and 441 samples from domestic pigs and wild boars, respectively. Samples were tested by real-time RT-PCR, and RVA-positive samples were genotyped in VP7 and VP4 segments. Our results report an RVA prevalence of 49.9% in domestic pigs and 9.3% in wild boars. Outstanding RVA genetic diversity was observed in VP7 and VP4 segments, especially in domestic pigs exhibiting a striking 23 different RVA combinations (G5P13 and G9P23 prevailed). Interspecies transmission events were numerous between domestic pigs and wild boars, sharing G3, G5, G6, G9, G11 and P13 genotypes. Furthermore, our data indicate that such transmission events involved even bovines (G6, P11) and, intriguingly, humans (G1P8). This study contributes to the basic knowledge that may be considered important for vaccine development and introduction, as a valuable and currently missing tool for efficient pig health management in the EU.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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.
Hepatitis E virus (HEV) can infect a wide range of domestic and wild animals, and the identification of new host species is reported successively worldwide. Nevertheless, its zoonotic potential and ...natural transmission, especially in wildlife remains unclear, primarily due to the discrete nature of HEV infections. Since the red fox (
) is the most widespread carnivore worldwide, and has been recognized as a potential HEV reservoir, its role as a potent host species is of increasing interest. Another wild canine species, the jackal （
）, is becoming more important within the same habitat as that of the red fox since its number and geographical distribution have been rapidly growing. Therefore, we have chosen these wild species to determine their potential role in the epidemiology and persistence of HEV in the wilderness. The main reason for this is the finding of HEV and a rather high HEV seroprevalence in wild boars sharing the same ecological niche as the wild canine species, as well as the risk of the spread of HEV through red foxes into the outskirts of cities, where possible indirect and even direct contact with people are not excluded. Therefore, our study aimed to investigate the possibility of natural HEV infection of free-living wild canines, by testing samples for the presence of HEV RNA and anti-HEV antibodies to gain better epidemiological knowledge of the disease. For this purpose, 692 red fox and 171 jackal muscle extracts and feces samples were tested. Neither HEV RNA nor anti-HEV antibodies were detected. Although HEV circulation was not detected in the tested samples, to our knowledge, these are the first results that include jackals as a growing and important omnivore wildlife species for the presence of HEV infection in Europe.
•Two new subgroups of bovine respiratory syncytial virus were observed in Croatia.•New subgroup VIII strains dominated in 2014–2016, replacing subgroup VII strains.•Subgroup II and new subgroup VIII ...strains co-circulated in Croatia.•Subgroups II and VII harbour unique mutations in G gene, allowing immune escape.•Intensive bovine vaccination resulted in emergence of new virus variants.
Bovine respiratory syncytial virus (BRSV) represents an important causative agent of respiratory tract disease in cattle. This study describes the genetic diversity of BRSV strains detected in beef cattle herds in Croatia during four consecutive years, from the end of 2011 to April 2016. Genetic diversity of circulating Croatian strains is reflected in their clustering within three different genetic subgroups. Analysis of representative BRSV G gene sequences revealed that infections in Croatia were caused by BRSV strains belonging to two new subgroups (VII and VIII identified herein for the first time). In 2014–2016, the subgroup VII strains were replaced with BRSV strains clustered in the previously unidentified subgroup VIII. Furthermore, co-circulation of subgroup II and new subgroup VIII strains in Croatia was recorded in the same time period. Sequences of Croatian BRSV strains within subgroups II and VII revealed unique mutations within an essential immunodominant region, demonstrating continuous evolution of viral mechanisms for immune escape.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Canine adipose-derived mesenchymal stem cells (cAD-MSCs) hold therapeutic promise due to their regenerative potential, particularly within their secretome. However, concerns arise regarding the ...impact of
cultivation necessitated for storing therapeutic doses, prompting this study to comprehensively explore the impact of
aging on gene expression and secretome composition.
The study involved collecting abdominal adipose tissue samples from nine healthy female dogs, from which cAD-MSCs were extracted and cultured. Stem cells were validated through trilineage differentiation assays and flow cytometry immunophenotyping. Gene expression profiling using RT-qPCR array, and cAD-MSCs secretome LC-MS/MS analysis, were conducted at passages 3 and 6 to reveal gene expression and protein composition alterations during
culture.
The results demonstrate that the gene expression and secretome composition of cAD-MSCs were impacted by
aging. Among many alterations in gene expression between two passages, two significant downregulations were noted in the MSC-associated PTPRC and IL10 genes. While the majority of proteins and their functional characteristics were shared between passages, the influence of cell aging on secretome composition is highlighted by 10% of proteins being distinctively expressed in each passage, along with 21 significant up- and downregulations. The functional attributes of proteins detected in passage 3 demonstrated a greater inclination towards supporting the regenerative capacity of cAD-MSCs. Moreover, proteins in passage 6 exhibited a noteworthy correlation with the blood coagulation pathway, suggesting an elevated likelihood of coagulation events. To the best of our knowledge, this study presents the first original perspective on the changes in secretome composition that occur when cAD-MSCs age
. Furthermore, it contributes to broadening the currently restricted knowledge base concerning the secretome of cAD-MSCs. In conclusion, our findings show that the regenerative potential of cAD-MSCs, as well as their secretome, may be compromised by
aging. Therefore, our study suggests a preference for earlier passages when considering these cells for therapeutic applications.
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