Simultaneous Detection of Antigen and Antibodies of African Swine Fever in a Novel Combo Lateral Flow Assay.

African swine fever (ASF) is a contagious disease of wild boar and domestic pigs notifiable to the World Organisation for Animal Health due to its high socio-economic impact. ASF is caused by the complex ASF virus (ASFV), and it can present different clinical manifestations that can be confused with other diseases; for this reason, laboratory testing is necessary for the proper diagnosis of clinically suspected animals. Despite the efforts put into it over decades, no treatment or safe vaccine is globally available, and disease control is based on early diagnosis and the implementation of strict biosecurity measures. In this context, rapid tests have the potential to accelerate and facilitate the identification of infected animals by giving fast on-site results. In this work, we improved the available point-of-care assays for the diagnosis of the disease by the development of a more specific antigen test and a more sensitive antibody test. This antibody detection test allowed for the earlier detection of infected animals than two commercial indirect ELISAs (statistically significant). Moreover, we developed a combined dual rapid test, unifying, in the same cassette, an antigen detection strip and an antibody detection strip. In this study, we confirmed that this combo approach is a useful tool for implementing rapid tests in the field since it increases the percentage of positive samples detected, even when PCR turns negative, while maintaining a good specificity.

Prevalence of SARS-CoV-2 in domestic cats (Felis catus) during COVID-19 pandemic in Latvia.

BACKGROUND: The causative agent of the COVID-19 pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is of zoonotic origin and has shown reverse zoonotic transmissibility. OBJECTIVES: The aim of this cross-sectional study was to investigate the serological and molecular prevalence of SARS-CoV-2 infection in the domestic cat (Felis catus) population from Latvia in natural conditions and subsequently perform viral genome analysis. METHODS: Oropharyngeal and rectal swabs and blood samples were collected from 273 domestic cats during the second wave of COVID-19 infection in Latvia. Molecular prevalence was determined by using reverse transcriptase-polymerase chain reaction (RT-PCR). Serum samples were analysed via double antigen enzyme-linked immunosorbent assay targeting the antibody against the nucleocapsid protein of SARS-CoV-2. Positive swab samples were analysed using whole viral genome sequencing and subsequent phylogenetic analysis of the whole genome sequencing data of the samples was performed. RESULTS: The overall SARS-CoV-2 RT-PCR positivity and seroprevalence was 1.1% (3/273) and 2.6% (7/273), respectively. The SARS-CoV-2 genome sequences from three RT-PCR positive cats were assigned to the three common lineages (PANGOLIN lineage S.1.; B.1.177.60. and B.1.1.7.) circulating in Latvia during the particular period of time. CONCLUSIONS: These findings indicate that feline infection with SARS-CoV-2 occurred during the second wave of the COVID-19 pandemic in Latvia, yet the overall prevalence was low. In addition, it seems like no special 'cat' pre-adaptations were necessary for successful infection of cats by the common lineages of SARS-CoV-2.

Reproduction and Productivity in Dairy Cattle after Abortions Both Related and Unrelated to Coxiella burnetii.

C. burnetii is a widespread pathogen, causing abortions and reproductive disorders in ruminants. The study aimed to evaluate animal reproductive capacity and productivity after abortion, related and unrelated to C. burnetii. We compared data about the abortion time, the outcome of the animals after an abortion, further reproduction, and productivity for C. burnetii-positive (n = 148) and C. burnetii-negative (n = 149) aborted dairy cows and heifers. C. burnetii-positive animals had a positive serological response or presence of C. burnetii DNA at the time of abortion. C. burnetii-positive animals had a significantly higher number of lactations at the time of abortion. However, in the other indicators, we observed no significant differences between the groups. Comparing indicators of all the aborted animals, we found that if animals started a new lactation after abortion, they had a significantly lower milk yield, lower fat, protein, and somatic cell counts (SCCs) in milk during the standard lactation for both primiparous and multiparous cows compared to herd averages in each group. Lower SCCs can be due to animals with a high SCC being culled earlier. We found an economic disadvantage to aborting, not only because of the loss of offspring, but also because of the high culling rate and lower productivity in both primiparous and multiparous cows.

Do Seropositive Wild Boars Pose a Risk for the Spread of African Swine Fever? Analysis of Field Data from Latvia and Lithuania.

In 2020, ASF occurred in wild boars throughout Latvia and Lithuania, and more than 21,500 animals were hunted and tested for the presence of the virus genome and antibodies in the framework of routine disease surveillance. The aim of our study was to re-examine hunted wild boars that tested positive for the antibodies and negative for the virus genome in the blood (n = 244) and to see if the virus genome can still be found in the bone marrow, as an indicator of virus persistence in the animal. Via this approach, we intended to answer the question of whether seropositive animals play a role in the spread of the disease. In total, 2 seropositive animals out of 244 were found to be positive for the ASF virus genome in the bone marrow. The results indicate that seropositive animals, which theoretically could also be virus shedders, can hardly be found in the field and thus do not play an epidemiological role regarding virus perpetuation, at least not in the wild boar populations we studied.

Genetic diversity and known virulence genes in Listeria innocua strains isolated from cattle abortions and farm environment.

Listeria innocua is considered as non-pathogenic bacteria living in an environment although several cases of immunocompromised humans and ruminant listeriosis infections have been reported. Previously, L. innocua was identified as a potential pathogen and virulence in association with L. monocytogenes PrfA dependent virulence (LIPI-1) gene cluster was demonstrated in hemolytic L. innocua. L. innocua usually considered non-pathogenic versus pathogenic L. monocytogenes and L. ivanovii because of the main virulence gene loss. There are limited studies and reports available about L. innocua-caused illness in cattle. A total of 18 STs were identified in cattle abortions while 17 STs in the farm environment with majority of STs were present in both abortions and environmental samples. Genome sequencing showed that in one farm identical L. innocua clones were represented in water, feed, soil, and faeces sample groups, suggesting that animals most likely through the faecal shedding may remain as the main source of L. innocua in a farm environment. Out of all L. innocua isolates PrfA-dependent virulence genes were not found in aborted foetuses isolates and environmental L. innocua isolate groups; however, in 20% of isolates a complete LIPI-3 pathogenicity island encoding listeriolysin S was identified. In this study, we demonstrated that genetically diverse L. innocua clones were widely distributed in cattle farm environment and certain isolates had a significant pathogenicity potential for cattle, thus causing adverse health effects, including abortions.

A practical guide for strategic and efficient sampling in African swine fever-affected pig farms.

In the case of African swine fever (ASF) outbreaks in pig farms, EU legislation requires a thorough epidemiological investigation to determine, among other tasks, the extent of infection in the affected farm. The main aim of this study was to implement a reliable sampling strategy to quickly obtain an overview of the extent of ASF virus spread in an affected pig farm. We developed and tested a three-step approach: (i) identification of sub-units within the affected farm, (ii) categorization of sub-units, and (iii) targeted selection of animals for testing. We used commercially available lateral flow devices (LFDs) to detect ASF antigen and antibodies under field conditions and compared them with routinely performed laboratory tests (qPCR, ELISA, IPT). The study was conducted in three commercial farms in Latvia that were affected by ASF in July 2020. One of the affected farms was relatively small with only 31 pigs, whereas the other two were large with 1800 and 9800 animals, respectively. The approach proved to be helpful and practical for efficient and reliably assess the ASF situation on the farm and to identify sub-units within a farm where infected animals are present and sub-units which might (still) be free of infection. This important epidemiological information helps to better estimate the high-risk period and to track the potential spread of infection outside the farm. It allows also to prioritize culling and, if appropriate, to pursue a partial culling strategy taking into account the absence of clinical signs, implemented biosecurity measures, quarantine and negative test results, among others. This might be of interest for large commercial farms where the infection was identified very early and has not yet spread widely. Due to its limited sensitivity, the antigen LFD test is useful for testing animals showing signs of disease.

A European-wide dataset to uncover adaptive traits of Listeria monocytogenes to diverse ecological niches.

Listeria monocytogenes (Lm) is a ubiquitous bacterium that causes listeriosis, a serious foodborne illness. In the nature-to-human transmission route, Lm can prosper in various ecological niches. Soil and decaying organic matter are its primary reservoirs. Certain clonal complexes (CCs) are over-represented in food production and represent a challenge to food safety. To gain new understanding of Lm adaptation mechanisms in food, the genetic background of strains found in animals and environment should be investigated in comparison to that of food strains. Twenty-one partners, including food, environment, veterinary and public health laboratories, constructed a dataset of 1484 genomes originating from Lm strains collected in 19 European countries. This dataset encompasses a large number of CCs occurring worldwide, covers many diverse habitats and is balanced between ecological compartments and geographic regions. The dataset presented here will contribute to improve our understanding of Lm ecology and should aid in the surveillance of Lm. This dataset provides a basis for the discovery of the genetic traits underlying Lm adaptation to different ecological niches.

Characterization and Genetic Diversity of Listeria monocytogenes Isolated from Cattle Abortions in Latvia, 2013-2018.

Listeria monocytogenes can cause disease in humans and in a wide range of animal species, especially in farm ruminants. The aim of the study was to determine the prevalence and genetic diversity of L. monocytogenes related to 1185 cattle abortion cases in Latvia during 2013-2018. The prevalence of L. monocytogenes among cattle abortions was 16.1% (191/1185). The seasonality of L. monocytogenes abortions was observed with significantly higher occurrence (p < 0.01) in spring (March-May). In 61.0% of the cases, the affected cattle were under four years of age. L. monocytogenes abortions were observed during the third (64.6%) and second (33.3%) trimesters of gestation. Overall, 27 different sequence types (ST) were detected, and four of them, ST29 (clonal complex, CC29), ST37 (CC37), ST451 (CC11) and ST7 (CC7), covered more than half of the L. monocytogenes isolates. Key virulence factors like the prfA-dependent virulence cluster and inlA, inlB were observed in all the analyzed isolates, but lntA, inlF, inlJ, vip were associated with individual sequence types. Our results confirmed that L. monocytogenes is the most important causative agent of cattle abortions in Latvia and more than 20 different STs were observed in L. monocytogenes abortions in cattle.

Prevalence, Genetic Diversity and Factors Associated with Distribution of Listeria monocytogenes and Other Listeria spp. in Cattle Farms in Latvia.

Listeria spp. is a diverse genus of Gram-positive bacteria commonly present in the environment while L. monocytogenes and L. ivanovii are well known human and ruminant pathogens. The aim of the present study was to reveal the prevalence and genetic diversity of L. monocytogenes and other Listeria spp. and to identify the factors related to the abundance of pathogen at cattle farms. A total of 521 animal and environmental samples from 27 meat and dairy cattle farms were investigated and the genetic diversity of L. monocytogenes isolates was studied with WGS. The prevalence of Listeria was 58.9%, while of L. monocytogenes it was -11%. The highest prevalence of L. monocytogenes was found in the environment-soil samples near to manure storage (93%), mixed feed from the feeding trough and hay (29%), water samples from farms drinking trough (28%) and cattle feces (28%). Clonal complexes (CC) of CC37 (30%), CC11 (20%) and CC18 (17%) (all IIa serogroup) were predominant L. monocytogenes clones. CC18, CC37 and CC8 were isolated from case farms and CC37, CC11 and CC18 from farms without listeriosis history. Only one hypervirulent CC4 (1%) was isolated from the case farm. Sequence types (STs) were not associated with the isolation source, except for ST7, which was significantly associated with soil (p < 0.05). The contamination of soil, feeding tables and troughs with L. monocytogenes was associated with an increased prevalence of L. monocytogenes at farms. Our study indicates the importance of hygienic practice in the prevention of the dissemination of L. monocytogenes in the cattle farm environment.

Effect of immunoglobulin G concentration in dairy cow colostrum and calf blood serum on Cryptosporidium spp. invasion in calves.

AIM: The research aimed to test the association between the level of immunoglobulin G (IgG) in bovine colostrum and calf blood serum and to evaluate its relation to Cryptosporidium spp. invasion in calves. MATERIALS AND METHODS: Fresh colostrum and fecal specimens from cows (n=114) as well as blood and fecal specimens from newborn calves (n=114) were collected in the dairy cattle farm. Investigated calves were separated from their mothers directly after birth and received 2 L of colostrum in two separate feedings within the first 24 h. Blood samples were taken from calves at the age of 2 days. Coprological samples were taken from calves at the age of 1, 10, and 15 days. Both colostrum and fecal samples from cows were taken on the 1st day after calf birth. Rectal fecal samples were collected separately from each calf and cow into plastic bags. The collected calf serum samples and bovine colostrum samples were tested for bovine IgG by competitive enzyme-linked immunosorbent assay kit bovine Ig. To record oocysts of Cryptosporidium spp. in feces, the flotation method was used. Binomial logistic regression was performed to ascertain the effects of IgG in bovine colostrum and calf blood serum on the likelihood of Cryptosporidium spp. infection in calves. RESULTS: The concentration of IgG in bovine colostrum was higher (70.7±26.6 g/L, mean±standard deviation) than that in calf blood serum (13.2±6.1 g/L); the statistically significant difference was 57.4 g/L (95% confidence interval, 52.4-62.4), t (124.872)=22.536, p<0.001. Mann-Whitney's U-test showed a significant difference between samples collected on days 10 and 15 of the experiment (U=1944, z=2.330, p=0.020). The higher number of oocysts in calf feces was recorded on day 15 (median=6.5) compared to day 10 (median=4). The prevalence of calf infection from days 10 to 15 increased from 26.3 to 45.6% and was at least 3 times higher than in cows. A statistically significant positive correlation was recorded between IgG concentration of cow colostrum and calf blood serum (r (114)=0.414, p=0.001), whereas a correlation between the concentration of IgG and the intensity of Cryptosporidium spp. infection was not recorded (p>0.05). The logistic regression model was not statistically significant (χ2(2)=0.013, p=0.99 (10 days) and χ2(2)=0.100, p=0.95 (15 days)). CONCLUSION: Mother passive transfer of immunity to the offspring through colostrum does not influence the susceptibility of calves to Cryptosporidium infestation.

Seroprevalence of Brucella suis in eastern Latvian wild boars (Sus scrofa).

Brucellosis due to Brucella suis biovar 2 is one of the most important endemic diseases in wild boar (Sus scrofa) populations in Europe. The aim of the present study was to determine the seroprevalence of brucellosis in wild boars in the eastern part of Latvia. Wild boars killed by hunters in the period from January to April 2015 (n = 877) and from March to April in 2016 (n = 167) were examined for antibodies against B. suis by the Rose Bengal test (RBT), a complement fixation test (CFT), and by enzyme-linked immunosorbent assays. In 2015, 199 samples (22.7%) were positive by RBT and/or CFT while 36 samples (21.6%) were seropositive in 2016. Of the Brucella seropositive samples from 2015 and 2016 (n = 235), 162 (68.9%) were also seropositive to Yersinia enterocolitica. Considering cross-reactivity of serological tests, the seroprevalence of B. suis biovar 2 exposure in wild boars in the eastern part of Latvia was calculated to 14.0% in 2015 and 9.6% in 2016. From selected seropositive samples (42 in 2015 and 36 in 2016) total DNA was extracted and analyzed with an IS711-based nested polymerase chain reaction (PCR) assay. Species and biovar identification was conducted for bacteria isolated in monoculture from PCR positive samples by species specific primers and Bruce-ladder multiplex PCR. Brucella suis biovar 2 was isolated from 12/20 samples in 2015 and 9/9 samples in 2016. The average seroprevalence was relatively low compared to that found in certain other European countries. Males and females had an equal level of seropositivity, but a positive age-trend was observed for both males and females.

Legionella pneumophila Seropositivity-Associated Factors in Latvian Blood Donors.

Continuous environmental exposure of humans to Legionella may induce immune responses and generation of antibodies. The aim of this study was to investigate the seroprevalence of Legionella pneumophila serogroups (SG) 1-6 in the general healthy population and identify the associated host-related and environmental risk factors. L. pneumophila SG 1-6 seroprevalence among a total of 2007 blood samples collected from healthy donors was 4.8%. Seroprevalence was higher in women (5.9%) than men (3.3%) and in areas with a larger number of inhabitants, ranging from 3.5% in rural regions to 6.8% in the capital, Riga. Blood samples from inhabitants of apartment buildings tested positive for L. pneumophila in more cases (5.8%) compared to those from inhabitants of single-family homes (2.7%). Residents of buildings with a municipal hot water supply system were more likely to be seropositive for L. pneumophila (OR = 3.16, 95% CI 1.26-7.91). Previous episodes of fever were additionally identified as a risk factor (OR = 2.42, 95% CI 1.43-4.1). In conclusion, centralized hot water supply, female gender and previous episodes of fever were determined as the main factors associated with L. pneumophila seropositivity in our study population.