Differential Infection Patterns and Recent Evolutionary Origins of Equine Hepaciviruses in Donkeys.

The hepatitis C virus (HCV) is a major human pathogen. Genetically related viruses in animals suggest a zoonotic origin of HCV. The closest relative of HCV is found in horses (termed equine hepacivirus [EqHV]). However, low EqHV genetic diversity implies relatively recent acquisition of EqHV by horses, making a derivation of HCV from EqHV unlikely. To unravel the EqHV evolutionary history within equid sister species, we analyzed 829 donkeys and 53 mules sampled in nine European, Asian, African, and American countries by molecular and serologic tools for EqHV infection. Antibodies were found in 278 animals (31.5%), and viral RNA was found in 3 animals (0.3%), all of which were simultaneously seropositive. A low RNA prevalence in spite of high seroprevalence suggests a predominance of acute infection, a possible difference from the mostly chronic hepacivirus infection pattern seen in horses and humans. Limitation of transmission due to short courses of infection may explain the existence of entirely seronegative groups of animals. Donkey and horse EqHV strains were paraphyletic and 97.5 to 98.2% identical in their translated polyprotein sequences, making virus/host cospeciation unlikely. Evolutionary reconstructions supported host switches of EqHV between horses and donkeys without the involvement of adaptive evolution. Global admixture of donkey and horse hepaciviruses was compatible with anthropogenic alterations of EqHV ecology. In summary, our findings do not support EqHV as the origin of the significantly more diversified HCV. Identification of a host system with predominantly acute hepacivirus infection may enable new insights into the chronic infection pattern associated with HCV. IMPORTANCE: The evolutionary origins of the human hepatitis C virus (HCV) are unclear. The closest animal-associated relative of HCV occurs in horses (equine hepacivirus [EqHV]). The low EqHV genetic diversity implies a relatively recent acquisition of EqHV by horses, limiting the time span for potential horse-to-human infections in the past. Horses are genetically related to donkeys, and EqHV may have cospeciated with these host species. Here, we investigated a large panel of donkeys from various countries using serologic and molecular tools. We found EqHV to be globally widespread in donkeys and identify potential differences in EqHV infection patterns, with donkeys potentially showing enhanced EqHV clearance compared to horses. We provide strong evidence against EqHV cospeciation and for its capability to switch hosts among equines. Differential hepacivirus infection patterns in horses and donkeys may enable new insights into the chronic infection pattern associated with HCV.

A Retrospective Study on the Seroprevalence of West Nile Virus Among Donkeys and Mules in Bulgaria.

Background: West Nile virus (WNV) infection, caused by a flavivirus, emerged in Europe and America in the past two decades. The etiological agent causes asymptomatic to life-threatening infection in humans and in some animal species. The objective of this study was to evaluate the seroprevalence of WNV among donkeys and mules in Bulgaria. Methods: A total of 200 archived serum samples were tested by competitive enzyme-linked immunosorbent assay (ELISA). Positive samples were additionally analyzed by virus neutralization assay. Results: Seroprevalence of 7% (14/200) was established among tested animals by ELISA. Two samples were subsequently verified for the presence of virus neutralizing antibodies; thus, the seroprevalence against WNV was determined to be 1% (2/200 [confidence interval = 0.12-3.61]). Positive results among mules included in the study were not found. Conclusion: The findings in the present research demonstrate that donkeys are exposed to WNV infection and seroconvert, which adds to the understanding of virus circulation among donkeys in settlements in north and south Bulgaria.

Seroprevalence of West Nile Virus among Equids in Bulgaria in 2022 and Assessment of Some Risk Factors.

The aim of this study was to analyze the seroprevalence of West Nile virus (WNV) among equids in Bulgaria, confirm the results of a competitive ELISA versus the virus neutralization test (VNT) and investigate some predisposing factors for WNV seropositivity. A total of 378 serum samples from 15 provinces in northern and southern Bulgaria were tested. The samples originated from 314 horses and 64 donkeys, 135 males and 243 females, aged from 1 to 30 years. IgG and IgM antibodies against WNV protein E were detected by ELISA. ELISA-positive samples were additionally tested via VNT for WNV and Usutu virus. Thirty-five samples were WNV-positive by ELISA (9.26% [CI = 6.45-12.88]), of which 15 were confirmed by VNT; hence, the seroprevalence was 3.97% (CI = 2.22-6.55). No virus-neutralizing antibodies to Usutu virus were detected among the 35 WNV-ELISA-positive equids in Bulgaria. When compared with VNT, ELISA showed 100.0% sensitivity and 94.5% specificity. A statistical analysis showed that the risk factors associated with WNV seropositivity were the region (p < 0.0001), altitude of the locality (p < 0.0001), type of housing (p < 0.0001) and breed (p = 0.0365). The results of the study demonstrate, albeit indirectly, that WNV circulates among equids in northern and southern Bulgaria, indicating that they could be suitable sentinel animals for predicting human cases and determining the risk in these areas or regions of the country.

Human ELISA Detects anti-SARS-CoV-2 Antibodies in Cats: Seroprevalence and Risk Factors for Virus Spread in Domestic and Stray Cats in Bulgaria.

The aim of this study was to verify whether the human DR-ELISA for the detection of anti-SARS-CoV-2 antibodies can be applied in cats, and to assess the risk factors that determine the spread of the virus among the cat population in Bulgaria. The study included 92 serum samples collected from 68 domestic and 24 stray cats aged from 3 months to 20 years of age in the period of January-June 2021. The samples originated from three regions in Bulgaria and from three places of inhabitance. DR-ELISA based on peroxidase-labeled SARS-CoV-2 N protein was employed to detect IgA, IgG and IgM antibodies in the samples. Subsequently, the results were compared with a commercially available multi-species ELISA kit. There was high seroprevalence (83.33%) in stray cats and 41.18% in domestic cats, confirmed by the human and veterinary ELISA kit. The positive cases in the regional cities were 42.86%, in small towns 50% and in villages 78.26%. Cats under 7 years had a five times higher risk than those over 7 years (p = 0.001). The risk was seven times higher for stray cats than for domestic cats (p = 0.001). In addition, the results indicate that the risk was the highest for cats in villages (p = 0.006) compared to cats in other places of inhabitance. This study demonstrates that human DR-ELISA may be successfully applied to monitor the circulation of SARS-CoV-2 in cats and other susceptible species. Cats might serve as sentinel animals for tracking the virus in nature and in inhabited areas (strays) and to discover asymptomatic cases in humans/owners.

Comparison between Some Phenotypic and Genotypic Methods for Assessment of Antimicrobial Resistance Trend of Bovine Mastitis Staphylococcus aureus Isolates from Bulgaria.

The aim of this study was to assess the resistance of bovine mastitis S. aureus isolates from farms in Bulgaria to different classes of chemotherapeutic drugs by comparison of some phenotypic and genotypic methods by means of Cohen's kappa statistics. The study comprised 546 milk samples from subclinical and clinical mastitis at 14 farms from 9 districts in the country. A total of 92 Staphylococcus aureus strains were isolated from tested samples and identified by nuc PCR. The results demonstrated high levels of resistance to sulfadimethoxine (87%), followed by resistance to penicillin (33.7%), erythromycin (13%), streptomycin (8.7%), tetracycline (6.5%) and gentamicin (1.1%). The comparison of both phenotypic tests with respect to 9 antimicrobials revealed strong agreement with kappa coefficient 0.836. An almost complete agreement was evidenced between phenotypic resistance to penicillin and blaZ gene presence, to methicillin with mecA gene, to tetracycline with tet genes, but the agreement between erythromycin resistance and erm genes presence was moderate. This study was the first to demonstrate discrepancy between the behaviour to cefoxitin in the disk diffusion test and oxacillin in the MIC test for an isolate shown to carry the mecA gene in the subsequent genetic analysis. Considering the detected discrepancies for some of isolates, an integral evaluation through phenotypic and molecular methods for monitoring of antimicrobial resistance of Staphylococcus aureus is recommended.

Population Pharmacokinetic Modelling of Orally Administered Doxycycline to Rabbits at Different Ages.

Doxycycline is a well-tolerated tetracycline antibiotic, registered for use in rabbits and administered for treatment of bacterial infections in this animal species. Nevertheless, the available pharmacokinetic data are limited and this study aimed to investigate the pharmacokinetics of orally administered doxycycline in mature and immature rabbits by application of the population approach. The rabbits were treated orally with doxycycline hyclate (5 mg/kg bw) in the form of a solid gelatin capsules. Free plasma concentrations were determined with HPLC analysis with Photodiode array detection. The estimated typical value of volume of distribution (tvV), total body clearance, and absorption rate constant were 4.429 L/kg, 1.473 L/kg/h, and 0.257 h-1, respectively. The highest between-subject variability (BSV) of 69.30% was observed for tvV. Co-variates such as body weight, age, and biochemical parameters did not improve the tested model and did not contribute to explanation of the BSV. The population pharmacokinetic model of the orally administered doxycycline in rabbits should be further developed by addition of data from more animals treated with higher doses. An oral dose of 5 mg/kg could ensure percentage of the time from the dosing interval during which the concentration is above minimum inhibitory concentration (MIC) %fT > MIC of 35% if MIC of 0.18 µg·mL-1 and a dosing interval of 12 h is assumed which does not cover criteria for rational use of antibiotics.

Effects of fluoroquinolone treatment on MDR1 and MRP2 mRNA expression in Escherichia coli-infected chickens.

Current knowledge about the expression of ABC transport proteins suggests that their expression is regulated by a variety of factors, including pathological conditions, and in particular inflammatory reactions to infection. As ABC transporters are major determinants of absorption, distribution and excretion of many antimicrobials, modulation of their activity may result in increased or decreased tissue levels of drugs, affecting the efficacy of treatment. As fluoroquinolones have been identified as modulators and substrates of a number of drug transporters, we evaluated the effect of danofloxacin mesylate and enrofloxacin treatment on the levels of expression of MDR1 and MRP2 mRNAs in the intestines and livers of broilers with experimentally induced colibacillosis. MDR1 mRNA expression was significantly decreased in infected animals and was partly restored over 5 days of treatment with orally administered danofloxacin mesylate or enrofloxacin. Changes in the level of expression of MRP2 mRNA were less prominent. The study suggests that the treatment of colibacillosis with fluoroquinolones, which resulted in a significant clinical improvement of the animals, also restored the expression of drug transporters. This is of clinical importance as these ABC transporters significantly contribute to the functionality of important biological barriers, protecting the bird and specific tissues from pathogens and bacterial toxins.