Wild boar (Sus scrofa) carcasses as an attraction for scavengers and a potential source for soil contamination with the African swine fever virus.

The wild boar (Sus scrofa) is a social animal species native to Eurasia. During the last decade, the wild boar population in Estonia has been severely affected by the African swine fever virus (ASFV), which has also affected domestic pig farming. The potential transmission routes of ASFV remain unclear and are currently under intensive investigation. This pilot study aimed to clarify the frequency and characteristics of contacts between living wild boars and the carcasses of their conspecifics, which could play a role in the transmission of ASFV. Wild animals' contact and scavenging behavior on wild boar carcasses were studied using trail cameras in an experimental setting on Hiiumaa, Western Estonia. Four legally hunted carcasses were used in the present study. This study aimed to determine whether intraspecies scavenging occurs in wild boars. The persistence of ASFV DNA in soil contaminated with infected wild boar carcasses was investigated separately. Among the 17 identified wildlife species that visited wild boar carcasses, the common raven (Corvus corax) was the most frequent one (37.26%), followed by raccoon dogs (Nyctereutes procyonoides; 4.25%), carcass conspecific/wild boars (3.16%), and red foxes (Vulpes vulpes; 2.14%). Regarding the direct contact with the carcass, the same species ranking was detected: common raven (74.95%), raccoon dogs (9.94%), wild boars (4.21%), and red foxes (4.21%). No clear signs of cannibalism were noted among the wild boars, although brief physical contact with the carcasses was evident. The persistence of ASFV DNA in soil contaminated by infected wild boar carcasses was investigated separately. This study revealed that ASFV DNA from infected carcasses could be detected in forest soil for prolonged periods, even after removing the carcasses. Hence, the carcasses of infected wild boars may play an important role in spreading the African swine fever virus in wild boar populations; thus, prompt removal and disinfection of the soil could be considered necessary to limit the spread of the infection.

Differentiation of African Swine Fever Virus Strains Isolated in Estonia by Multiple Genetic Markers.

The African swine fever virus (ASFV) was first detected in Estonia, in September 2014. In the subsequent three years, the virus spread explosively all over the country. Only one county, the island of Hiiumaa, remained free of the disease. Due to the drastic decrease in the wild boar population in the period of 2015-2018, the number of ASFV-positive cases among wild boar decreased substantially. From the beginning of 2019 to the autumn of 2020, no ASFV-positive wild boar or domestic pigs were detected in Estonia. A new occurrence of ASFV was detected in August 2020, and by the end of 2022, ASFV had been confirmed in seven counties in Estonia. Investigations into proven molecular markers, such as IGR I73R/I329L, MGF505-5R, K145R, O174L, and B602L, were performed with the aim of clarifying whether these cases of ASFV were new entries or remnants of previous epidemics. The sequences from the period of 2014-2022 were compared to the Georgia 2007/1 reference sequence and the variant strains present in Europe. The results indicated that not all the molecular markers of the virus successfully used in other geographical regions were suitable for tracing the spread of ASFV in Estonia. Only the B602L-gene analysis enabled us to place the ASFV isolates spreading in 2020-2022 into two epidemiologically different clusters.

Coxiella burnetii Seroprevalence and Associated Risk Factors in Cattle, Sheep, and Goats in Estonia.

Q fever, a disease caused by Coxiella burnetii (CB), is an emerging zoonotic health problem. The prevalence data from potential sources are valuable for assessing the risk to human and animal health. To estimate the prevalence of CB antibodies in Estonian ruminants, pooled milk and serum samples from cattle (Bos taurus) and pooled serum samples from sheep (Ovis aries) and goats (Capra hircus) were analyzed. Additionally, bulk tank milk samples (BTM; n = 72) were analyzed for the presence of CB DNA. Questionnaires and herd-level datasets were used to identify the risk factors for exposure using binary logistic regression analysis. The prevalence of CB-positive dairy cattle herds (27.16%) was significantly higher than that in beef cattle herds (6.67%) and sheep flocks (2.35%). No CB antibodies were detected in the goat flocks. CB DNA was found in 11.36% of the BTM samples. The odds of seropositivity were higher in dairy cattle herds, with an increasing number of cattle in the herd, and with location in southwestern, northeastern and northwestern Estonia. Dairy cattle herds had higher odds of testing positive for CB in BTM if the dairy cows were kept loose and lower odds if the herd was located in northwestern Estonia.

Effect of colostrum on the acute-phase response in neonatal dairy calves.

The core part of the mammal innate immune system is the acute-phase response (APR), during which acute-phase proteins (APP) are synthesized. Colostrum contains immunomodulating factors such as proinflammatory cytokines and APP in large quantities. We looked at proinflammatory cytokines [IL-1ß, IL-6, and tumor necrosis factor-α (TNF-α)] and APP [serum amyloid A (SAA) and haptoglobin (Hp)] in colostrum and in calves' serum. The aim of this study was to evaluate the effects of colostrum on the calves' systemic APR and the associations of the calves' serum APR with short- and long-term weight gain (at the age of 1, 3, and 9 mo). A total of 143 female dairy calves were studied during their first 3 wk of life. The calves were separated from their mothers immediately after birth and bottle-fed 3 L of quality-controlled colostrum once within 2 h after birth. Serum samples were collected once a week during the first 3 wk of life (a total of 1-3 samples per calf). Mean sampling age (±standard deviation) was 4.3 (±2.0) d in the first week, 11.0 (±2.0) d in the second week, and 18.0 (±2.0) d in the third week. Linear regression models were used to study associations of colostrum APP and cytokine concentration with serum APR markers and for studying associations of colostrum and serum APR markers with calves' average daily weight gain (ADWG). Mixed linear regression models were used to compare serum concentrations of APR markers by study weeks. The colostrum IL-6 concentrations were positively associated with serum IL-6 in the first 3 wk of life. Colostrum IL-1ß was positively associated with calves' serum IL-1ß during the first week of life, and colostrum TNF-α was positively associated with calves' serum TNF-α during the first 2 wk of life. Serum IL-1ß concentrations differed over the 3 wk, being the highest during the first week and the lowest during the second week. For IL-6, the concentration during the first week was the highest, and for TNF-α, a steady decline in the concentration was observed. Serum SAA concentrations were elevated during the first 2 wk of life and subsequently declined during the third week. Albumin concentrations were lowest in the first week, whereas Hp concentrations were highest during the second week. Serum concentrations of SAA, Hp, IL-6, and TNF-α during the second week were negatively associated with ADWG at 9 mo of age. The SAA concentrations during the third week of age had a negative association with 9-mo ADWG. Serum Hp concentrations in the third week were negatively associated with 3-mo ADWG. The results of our study suggest that colostrum cytokines influence calf serum cytokine concentrations. Thus, they influence the newborn calves' adaptation to the environment and the development of their immune system. Factors that activate an APR during the second and third week of life have a long-term influence on calves' development.

Rediscovering the mosquito fauna (Diptera: Culicidae) of Estonia: an annotated checklist with distribution maps and DNA evidence.

Female mosquitoes (Diptera: Culicidae) affect their hosts in numerous negative ways and are crucial to the spread of vector-borne pathogens. It is, therefore, important to have a detailed overview of regional mosquitoes, to be able to detect changes in species diversity and identify possible health threats. The aim of this study was to update the checklist of the mosquito fauna of Estonia for the first time since 1957. For this purpose, 24,344 adult mosquitoes (94% females) were collected in Estonia from 2008 to 2020 using various trapping methods. Specimens were primarily identified by morphological characteristics, but DNA barcoding based on the partial cytochrome c oxidase subunit 1 gene (COI) was also used. Species were included in the checklist based on historical records as well as new collections, while also considering reports from neighboring countries. Species records are supplemented with voucher specimens, distribution maps and DNA evidence. The updated checklist includes 34 species, 27 of which were confirmed with recently collected material. All in all, Aedes communis (de Geer, 1776) proved to be the most common mosquito in Estonia, accounting for 30.1% of the specimens collected. This is noteworthy, as this species has been implicated in the transmission of multiple disease agents present in the area. New evidence revealed the presence of Ae. hexodontus Dyar, 1916, Ae. sticticus (Meigen, 1838), Anopheles messeae Falleroni, 1926 and Culiseta bergrothi (Edwards, 1921) in Estonia.

Variation in the Distribution of Nosema Species in Honeybees (Apis mellifera Linnaeus) between the Neighboring Countries Estonia and Latvia.

The unicellular spore-forming parasites Nosema apis and Nosema ceranae are considered to be one of the causes of increased honey bee mortality in recent years. These pathogens attack their honey bee hosts through their gut, causing changes in behavioral stress responses and possibly resulting in decreased honey yield and increased honey bee mortality. The present study aimed to determine the prevalence of Nosema spp. (nosemosis) in Estonia and Latvia, as well as the persistence of the disease in previously infected hives. Currently, N. ceranae is considered the most virulent species and is predominant worldwide. However, in some regions, usually with colder climates, N. apis is still prevalent. To achieve better disease control, it is important to determine the species distribution. For this purpose, we selected 30 apiaries in Estonia and 60 in Latvia that were positive for Nosema spp. in the EPILOBEE (2012-2014) study, which was 5 years prior to the present study. The results show that, while both species are present in Estonia and Latvia, N. apis is dominant in Estonia (43%), and N. ceranae is dominant in Latvia (47%). We also found that the pathogens are very persistent, since 5 years later, only 33% of infected apiaries in Estonia and 20% of infected apiaries in Latvia, we could not detect any pathogens at the time of sampling.

Friends in All the Green Spaces: Weather Dependent Changes in Urban Mosquito (Diptera: Culicidae) Abundance and Diversity.

Mosquitoes (Diptera: Culicidae) are universally recognized as troublesome pests and vectors of various pathogens and parasites. Understandably, the species makeup and diversity of individual populations depends on local and broad scale environmental trends, especially on temperature and hydrological variations. Anthropogenic landscapes make for unique habitats, but their effect on insects likely varies across climatic regions. The aim of this study was to investigate the diversity and seasonal patterns of urban mosquitoes in the boreal region. Specimens were collected with an insect net from May to September during three years and determined to species or species group level. Weather information was added to each data point and results analyzed using multivariate regression models. Fieldwork yielded 1890 mosquitoes from four genera. Both abundance and the effective number of species (ENS) significantly decreased during the study period. The number of collected mosquitoes had a negative correlation with wind speed and temperature, latter of which exhibited a negative association with humidity. Species succession followed predictable patterns, but with some variation between years. Still, Culex pipiens/Culex torrentium were the most abundant throughout the study. Importantly, all dominant species were known disease vectors. Our work showed that higher temperatures could result in fewer mosquitoes in boreal towns.

No evidence for African swine fever virus DNA in haematophagous arthropods collected at wild boar baiting sites in Estonia.

African swine fever (ASF) is a highly pathogenic viral disease affecting all Suidae, with Ornithodoros moubata complex soft ticks acting as the biological arthropod vectors of the causative agent, African swine fever virus (ASFV). While ASFV is also transmissible via direct contact, pig products and fomites, other arthropods may be involved in virus transmission and persistence. Therefore, we checked various groups of blood-feeding arthropods collected during summer 2017 in wild boar habitats on the Estonian Island of Saaremaa for the presence of ASFV. Saaremaa had the highest ASF infection prevalences in Estonia in 2017, with an incidence of 9% among hunted wild boar. In addition to ASFV, we tested for other selected pathogens. In total, 784 ticks, 6,274 culicoid biting midges, 77 tabanids and 757 mosquitoes were tested as individuals or pools. No ASFV-DNA was found in any of them although about 20% of the tick samples tested positive for swine DNA. By contrast, tick-borne encephalitis virus-RNA was detected in one out of 37 tick pools (2.7%) and Borrelia burgdorferi s.l.-DNA in 20 individual ticks and 17 tick pools (25.2% of all samples). No Schmallenberg virus was detected in the Culicoides specimens. In conclusion, we found no evidence for Ixodes ricinus ticks, Culicoides punctatus and Obsoletus complex biting midges, Aedes spp., Anopheles spp. and Culiseta annulata mosquitoes, and Haematopota pluvialis tabanids playing a role in ASFV transmission in the wild boar population in Estonia.

Novel Mitochondrial DNA Lineage Found among Ochlerotatus communis (De Geer, 1776) of the Nordic-Baltic Region.

The Ochlerotatus (Oc.) communis complex consist of three Northern American species as well as a common Holarctic mosquito (Diptera: Culicidae) Oc. communis (De Geer, 1776). These sister species exhibit important ecological differences and are capable of transmitting various pathogens, but cannot always be differentiated by morphological traits. To investigate the Oc. communis complex in Europe, we compared three molecular markers (COI, ND5 and ITS2) from 54 Estonian mosquitoes as well as two COI marker sequences from Sweden. These sequences were subjected to phylogenetic analysis and screened for Wolbachia Hertig and Wolbach symbionts. Within and between groups, distances were calculated for each marker to better understand the relationships among individuals. Results demonstrate that a group of samples, extracted from adult female mosquitoes matching the morphology of Oc. communis, show a marked difference from the main species when comparing the mitochondrial markers COI and ND5. However, there is no variance between the same specimens when considering the nuclear ITS2. We conclude that Oc. communis encompasses two distinct mitochondrial DNA lineages in the Nordic-Baltic region. Further research is needed to investigate the origin and extent of these genetic differences.

Diversity of Diptera Species in Estonian Pig Farms.

In light of the African swine fever outbreaks in Estonian pig farms during the past few years, the question of the vector potential of Diptera in the pig farm environment has risen. However, the arthropod fauna of the pig farm environment is currently not well established. Hence, the aim of this study was to clarify the species diversity in pig farms. In total, 22 Diptera species or species groups were found in Estonian pig farms. There were altogether 186,701 individual arthropods collected, from which 96.6% (180,444) belonged to the order of true flies (Insecta: Diptera). The remaining 3.4% were from other insect orders, arachnids, or just damaged and unidentifiable specimens. The activity density and diversity of dipterans differed significantly between 12 sampled farms but not throughout the sampling period. The present study is amongst the few to provide a large-scale overview of pig-farm-associated Diptera in the temperate climate zone.

Trace amounts of African swine fever virus DNA detected in insects collected from an infected pig farm in Estonia.

BACKGROUND: African swine fever (ASF), a severe multi-systemic disease in pigs, was introduced into Estonia in 2014. The majority of outbreaks have occurred during the summer months. Given that ASFV is transmitted in a sylvatic cycle that includes the transmission by African soft ticks and that mechanical transmission by flying insects was shown, transmission by other arthropod vectors need to be considered. OBJECTIVES: Here, we report the results of a pilot study on flying insects caught on an outbreak farm during epidemiological investigations. METHODS: In brief, 15 different insect species (flies and mosquitoes) were collected by random catch using an aerial net. Nucleic acids derived from these samples or their pools were tested for African swine fever virus (ASFV) DNA by real-time PCR. RESULTS AND CONCLUSIONS: Viral DNA was detected in small quantities in two samples from flies and mosquitoes. Given the slow spread of virus within the farm, the impact of these findings seems rather low, but a role in local transmission cannot be ruled out. However, given the very low number of insects sampled, and taken into the account that viral isolation was not performed and insects outside the farm were not investigated, future investigations are needed to assess the true impact of insects as mechanical vectors.

Molecular epidemiology of Cryptosporidium spp. in calves in Estonia: high prevalence of Cryptosporidium parvum shedding and 10 subtypes identified.

We investigated the molecular epidemiology of Cryptosporidium spp. in Estonia by testing fecal samples from 486 calves aged <2 months, raised on 53 cattle farms, for the presence of Cryptosporidium DNA. The parasites were identified and characterized by sequencing of the 18S rRNA gene and of the 60 kDa glycoprotein (gp60) gene. Moreover, using a questionnaire, we surveyed factors that could be relevant for animal-to-human and human-to-animal transmission of Cryptosporidium spp. on the farms. Cryptosporidium spp. were shed by 23% of the investigated calves and at least one shedding calf was found on 66% of the farms. Cryptosporidium parvum was the most common species shed, while C. bovis and C. ryanae were also detected. More than half of the calves aged 8-14 days shed C. parvum. Nine previously described C. parvum subtypes (IIaA14G1R1, IIaA16G1R1, IIaA17G1R1, IIaA18G1R1, IIaA19G1R1, IIaA20G1R1, IIaA21G1R1, IIaA22G1R1 and IIaA16G2R1) and an apparently novel subtype IIlA21R2 were found. Calves from farms that reported spreading manure on fields during spring had 10 times higher odds to shed Cryptosporidium spp. in their feces than calves from farms that did not. Calves aged 8-14 days had higher odds to shed IIa18G1R1 as well as IIaA16G1R1 than younger calves.

Acute phase response in organic lambs associated with colostrum serum amyloid A, weight gain, and Cryptosporidium and Giardia infections.

In neonatal period, lamb's immune system goes through rapid adaptation to the extra-uterine environment. Success of this process can influence the animal's future performance and, thus, the quantitative assessment of it would greatly benefit sheep producers. The current study was conducted to investigate the acute phase response (APR) (measured through serum amyloid A (SAA), haptoglobin (Hp) and albumin (ALB)) in relation to later life growth (measured at 122 days of age), and naturally occurring Cryptosporidium and Giardia infections in neonatal lambs grown in organic farm. Serum (n = 692) and faecal (n = 141) samples were collected from 269 lambs in their first 3 weeks of life. The ewes' colostrum (n = 181) SAA concentrations were positively associated with the lambs' serum SAA and Hp concentrations at 2 to 4 days of age. Hp and ALB concentrations at the second week of age were positively associated with the growth rate at 122 days of age. Lamb serum globulin (GLOB) concentrations and Cryptosporidium-positive faecal samples were negatively associated at the second and third weeks of life. These findings suggest the importance of interactions between the immune system and environmental factors at the second week of the lambs' lives and its association with future performance.

Prevalence of cysticercosis in Estonian pigs and cattle.

Taenia solium has been ranked as the most important foodborne parasite and Taenia saginata as the most commonly found human Taenia tapeworm worldwide. The last official reports of taeniosis from Estonia were in 2003 for T. solium and 2012 for T. saginata. By law, all animal cases of cysticercosis must be registered and reported when found. Our aim was to estimate the prevalence of cysticercosis in Estonia caused by T. solium in pigs and T. saginata in cattle. The four slaughterhouses participating in the study slaughter between them approximately 80% of pigs and cattle in Estonia annually. Sampling spanned from February to April 2014, visiting the slaughterhouses five times per week. Visual inspection, palpation, and incisions at predilection sites were used to find cysts in both species. The sites inspected in both species were the external masseter, tongue, heart, and diaphragm. In addition, the internal masseter in pigs was examined, and the internal pterygoid muscle and esophagus in cattle. DNA was extracted from the cysts and used for PCR amplification of the cox1-gene for Taenia genus and species identification. A total of 564 cattle and 1217 pigs were examined. Cysts were found in 0.36% (n = 2; CI 0.06-1.17) of cattle and in 0.08% (n = 1; CI 0.004-0.40) of pigs. Cestode PCR was negative from all cysts. Results should be considered taking into account the low sensitivity and specificity of finding cysts. Results reflect the situation in larger slaughterhouses, and the possibility that the situation in smaller slaughterhouses is different should not be excluded.

Serological Evidence of Exposure to Globally Relevant Zoonotic Parasites in the Estonian Population.

We investigated Estonian population and its selected subgroups for serological evidence of exposure to Ascaris lumbricoides, Echinococcus spp., Taenia solium, Toxocara canis, Toxoplasma gondii, and Trichinella spiralis. Serum samples from 999 adults representing general population, 248 children aged 14-18, 158 veterinarians, 375 animal caretakers, and 144 hunters were tested for specific immunoglobulin G antibodies against the selected parasites using commercial enzyme immunoassays (ELISA). Sera yielding positive or twice grey zone Echinococcus spp, T. solium, T. canis, and T. spiralis results were subjected to western blot (WB) analysis. In the general population, based on the ELISA results, the A. lumbricoides seroprevalence was 12.7%, Echinococcus spp. seroprevalence was 3.3%, T. solium seroprevalence was 0.7%, T. canis seroprevalence was 12.1%, T. gondii seroprevalence was 55.8%, and T. spiralis seroprevalence was 3.1%. Ascaris lumbricoides seroprevalences were higher in children and in animal caretakers than in the general population, and T. canis seroprevalence was higher in animal caretakers than in the general population. Compared with the general population, Echinococcus spp. seroprevalence was higher in children. By contrast, T. gondii seroprevalence was higher in animal caretakers, and lower in children, than in the general population. In the general population, the WB-confirmed Echinococcus spp. seroprevalence was 0.5%, T. solium cysticercosis seroprevalence was 0.0%, Toxocara spp. seroprevalence was 14.5%, and Trichinella spp. seroprevalence was 2.7%. WB-confirmed Toxocara spp. seroprevalence was higher in animal caretakers than in the general population. We found serological evidence of exposure to zoonotic parasites in all tested groups. This calls for higher awareness of zoonotic parasitic infections in Estonia.

Characterization of the vaginal micro- and mycobiome in asymptomatic reproductive-age Estonian women.

The application of high-throughput sequencing methods has raised doubt in the concept of the uniform healthy vaginal microbiota consisting predominantly of lactobacilli by revealing the existence of more variable bacterial community composition. As this needs to be analyzed more extensively and there is little straightforward data regarding the vaginal mycobiome of asymptomatic women we aimed to define bacterial and fungal communities in vaginal samples from 494 asymptomatic, reproductive-age Estonian women. The composition of the vaginal microbiota was determined by amplifying bacterial 16S rRNA and fungal internal transcribed spacer-1 (ITS-1) regions and subsequently sequencing them using 454 Life Sciences pyrosequencing. We delineated five major bacterial community groups with distinctive diversity and species composition. Lactobacilli were among the most abundant bacteria in all groups, but also members of genus Gardnerella had high relative abundance in some of the groups. Microbial diversity increased with higher vaginal pH values, and was also higher when a malodorous discharge was present, indicating that some of the women who consider themselves healthy may potentially have asymptomatic bacterial vaginosis (BV). Our study is the first of its kind to analyze the mycobiome that colonizes the healthy vaginal environment using barcoded pyrosequencing technology. We observed 196 fungal operational taxonomic units (OTUs), including 16 OTUs of Candida spp., which is more diverse than previously recognized. However, assessing true fungal diversity was complicated because of the problems regarding the possible air-borne contamination and bioinformatics used for identification of fungal taxons as significant proportion of fungal sequences were assigned to unspecified OTUs.

Antioxidant protection and plasma carotenoids of incubating great tits (Parus major L.) in relation to health state and breeding conditions.

Carotenoids are biologically active pigments, which are important for animals due to their dual role in health maintenance and ornamental signalling. In adult birds, immunostimulatory properties of carotenoids have been repeatedly demonstrated while much less is known about the importance of carotenoids as antioxidants. We studied the relationships between plasma carotenoid levels, as well as total antioxidant protection, and various hemato-serological health state indices in female great tits (Parus major L.), incubating their second clutches in two contrasting (coniferous and deciduous) habitats in southwest Estonia. To manipulate reproductive effort, four eggs were removed from half of the clutches during laying to stimulate females to lay additional eggs. However, egg removal had no effect on the final number of eggs laid. Plasma carotenoid levels increased seasonally in parallel with caterpillar food availability. However, no between-habitat differences in carotenoid levels, total antioxidant capacity, or indices of health state could be found despite the apparently better feeding conditions in the coniferous habitat. No correlation was detected between plasma carotenoid levels and measures of total antioxidant capacity, which suggests that at least for the adult birds feeding on naturally carotenoid-rich diet, antioxidant function of carotenoids is not of primary importance. A strong non-linear association between the measures of antioxidant protection and leukocytic markers of inflammation was found, which suggests that measures of total antioxidant capacity deserve further attention in ecophysiological studies as potential indicators of immunopathology.

Predictors and markers of resistance to neurotropic nematode infection in rodent host.

Parasite-mediated selection is currently believed to play an important role in life-history evolution. To assess how simple hematoserological and biochemical condition indices reflect host immunocompetence and infection resistance controlled laboratory experiments are required. We addressed these issues by infecting laboratory rats with the standard dose of embryonated eggs of a neurotropic nematode Toxocara canis. Urine baseline corticosterone concentrations, measured 1 week before infection, predicted the number of nematode larvae later recovered from host brains. Thus, this noninvasive clinical marker appeared useful for assessment of potential infection resistance. Rats who had accumulated high number of larvae in their brains and muscle had large spleens and high peripheral eosinophil counts 17 days postinfection. This finding is consistent with the concept that induction of eosinophilic Th2 type humoral immune response benefits the parasite rather than host. Hence, excessive peripheral eosinophilia and spleen enlargement are not markers of efficient antiparasite response in larval toxocariasis.