Science is still searching for readily available, cost-effective biomarkers to assess metabolic disorders occurring before the onset and during the development of type-2 diabetes (T2DM). The aim of the present study was to induce T2DM in rats through a high-fat diet, followed by a single administration of low dose streptozotocin (STZ), and make an assessment of the development of the disease. The rats were divided into two groups—experimental and control—and were monitored for a period of 10 days. Changes in anthropometric parameters, glucose, insulin, lipids, uric acid, advanced oxidation protein products (AOPP), as well as the histological changes in the liver and pancreas, were recorded. To assess insulin resistance, we used the Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) and beta cell function (HOMA-β) and visceral obesity—adiposity index (AI). The data demonstrate that the increasing values of glucose, HOMA-IR, AI, total cholesterol, triacylglycerols, low- and very-low-density lipoproteins are important markers of the pre-diabetic state. The stable hyperglycemia and increased levels of TC, TG, VLDL, LDL, uric acid and AOPP in experimental rats strongly suggest the development of T2DM. HOMA-IR, HOMA-β, AI, and uric acid are reliable criteria for T2DM in rats.

The East Balkan swine (Sus scrofa) is the only aboriginal pig breed in Bulgaria, and it is indigenous to the eastern part of the country. The aim of the present study was to investigate East Balkan swine (EBS) in Bulgaria for serological evidence of hepatitis E virus (HEV). Sera from 171 swine from two parts of the country (northeastern and southeastern) were tested for anti-HEV IgG antibodies. The overall HEV seroprevalence was 82.5% (141/171), and for weaners it was 77.2% (44/57), for fattening pigs 79.0% (45/57), and for adults 91.2% (52/57). HEV positivity was higher in fattening pigs and adults compared to weaners: OR = 1.108 (95% CI: 0.456–2.692) and OR = 3.073 (95% CI: 1.016–9.294), respectively. This study provides the first evidence of exposure to HEV in EBS from Bulgaria.

In recent years, hepatitis E virus (HEV) infection has been found to be widespread among different animal species worldwide. In Bulgaria, high HEV seropositivity was found among pigs (60.3%), wild boars (40.8%), and East Balkan swine (82.5%). The aim of the present study was to establish the seroprevalence of HEV among dogs, cats, horses, cattle, sheep, and goats in Bulgaria. In total, 720 serum samples from six animal species were randomly collected: dogs—90 samples; cats—90; horses—180; cattle—180; sheep—90; and goats—90. The serum samples were collected from seven districts of the country: Burgas, Kardzhali, Pazardzhik, Plovdiv, Sliven, Smolyan, and Stara Zagora. The animal serum samples were tested for HEV antibodies using the commercial Wantai HEV-Ab ELISA kit (Beijing, China). The overall HEV seroprevalence among different animal species from Bulgaria was as follows: dogs—21.1%; cats—17.7%; horses—8.3%; cattle—7.7%; sheep—32.2%; and goats—24.4%. We found the lowest overall HEV seropositivity in Plovdiv district (6.2%; 4/64; p = 0.203) and Smolyan district (8.8%; 4/45; p = 0.129), vs. the highest in Pazardzhik district (21.6%; 29/134; p = 0.024) and Burgas district (28.8%; 26/90; p = 0.062). To the best of our knowledge, this is the first serological evidence of HEV infection in dogs, cats, horses, cattle, sheep, and goats from Bulgaria. We found high HEV seropositivity in small ruminants (sheep and goats), moderate seropositivity in pets (dogs and cats), and a low level of seropositivity in large animals (horses and cattle). Previous Bulgarian studies and the results of this research show that HEV infection is widespread among animals in our country. In this regard, the Bulgarian health authorities must carry out increased surveillance and control of HEV infection among animals in Bulgaria.

Background: Canine monocytic ehrlichiosis (CME) and canine granulocytic anaplasmosis (CGA), caused by Ehrlichia canis and Anaplasma phagocytophilum, are prevalent tick-borne diseases in dogs. Both trigger systemic inflammation, with C-reactive protein (CRP) and tumor necrosis factor-alpha (TNF-α) as potential biomarkers of severity. Aim: To compare serum CRP and TNF-α in CME, CGA, and co-infected dogs, and to assess their relationships with hematological, biochemical, and liver enzyme changes. Methods: In 134 dogs showing clinical signs of CME/CGA, infections were confirmed using SNAP® 4Dx® Plus, indirect immunofluorescence assay, and PCR. CRP and TNF-α were quantified using canine-specific ELISA kits. Results: Of the dogs tested, 112 (83.6%) were seropositive and 77 (68.8%) PCR positive (E. canis: 27; A. phagocytophilum: 29; co-infection: 21). Co-infected group had the highest CRP (135.2–154.8 mg/L) and TNF-α (161.5–174.0 pg/mL), significantly exceeding in CME (104.6–120.9 mg/L; 148.7–162.2 pg/mL), CGA (88.5–104.4 mg/L; 140.1–156.5 pg/mL), PCR-negative (33.1–45.8 mg/L; 12.3–18.9 pg/mL) and control groups (0.9–4.0 mg/L;0.2–2.3 pg/mL) with p < 0.001. Thrombocytopenia was common in all infected groups, with the lowest platelet counts in co-infected dogs (median 106.0 ×10⁹/L, p < 0.001). ALT and AST were significantly elevated only in co-infections (ALT: 89.25 U/L; AST: 69.38 U/L; p < 0.001). CRP correlated moderately with ALT/AST; TNF-α showed weaker positive associations. Conclusion: CRP and TNF-α are valuable indicators of systemic inflammation in CME and CGA, with maximal increases and stronger links to liver injury in co-infections, supporting their use in diagnosis and prognosis.

Infection of dogs with the tick-borne rickettsiae Ehrlichia and Anaplasma provokes an immune response mediating the pathology and bacterial resistance. IL-10 is the main anti-inflammatory cytokine and plays a multifaceted role in host protection. The study aimed to investigate circulating IL-10 in 32 dogs naturally infected with A. phagocytophilum and E. canis, identified by PCR positivity, and 33 PCR-negative animals which tested positive for antibodies against these pathogens, as well as 22 healthy animals. The highest quantity of IL-10, measured by ELISA, was observed among dogs positive simultaneously for anti-E. canis and anti-A. phagocytophilum IgG antibodies, followed by dogs positive for anti-E. canis only. The concentration of IL-10 in PCR-positive dogs was almost three and a half times higher than that measured in the control group (77.09 ± 23.61 pg./mL vs. 21.55 ± 4.61 pg./mL; p = 0.0015) and five times higher than the concentration of interleukin in PCR-negative animals (77.09 ± 23.61 pg./mL vs. 14.86 ± 3.01 pg./mL; p = 0.000016). The highest level of IL-10 was observed in PCR-positive dogs with mixed infection (120.54 ± 44.18), followed by the level in PCR-positive dogs for E. canis only (78.81 ± 16.92). The lowest level of IL-10 was observed in PCR-positive dogs for A. phagocytophilum only (56.32 ± 12.68). We may suggest that infection with E. canis and A. phagocytophilum stimulates the IL-10 production in dogs, which may facilitate specific antibody responses.

The protective effects of herbal feed additives Withania somnifera, Silybum marianum, Centella asiatica, and silymarin (administered at feed levels of 4000 ppm, 5000 ppm, 4600 ppm, and 25,000 ppm, respectively) against the toxic effects of ochratoxin A (OTA) (administered at 2 ppm) were investigated in 48 New Zealand White rabbits (37 days old) over an 80-day experimental period. A decrease in body weight was seen in rabbits exposed to OTA alone, but that decrease was less pronounced in rabbits receiving herbal supplements. The most severe lesions in OTA-treated rabbits were found in the liver, kidneys and spleen, while milder lesions were seen in the heart, intestine and lungs. The intensity of macroscopic, histopathological and biochemical changes was highest in rabbits exposed to OTA alone, followed by rabbits additionally supplemented with herbal additives, as evidenced by histopathological findings and serum levels of blood urea nitrogen (BUN), creatinine, aspartate aminotransferase (AST), and alanine aminotransferase (ALT). The most pronounced protective effects of the herbal additives were observed in the kidneys and liver. Oxidative stress was significantly increased in rabbits exposed to OTA alone, as evidenced by elevated reactive oxygen species (ROS), nitric oxide (●NO), ascorbyl radicals (●Asc), protein oxidation (PO), procollagen type I alpha 1 (COL1A1), malondialdehyde (MDA), kidney injury molecule-1 (KIM-1), hydroxyproline (Hyp), advanced glycation end products (AEGs), heme oxygenase-1 (HO-1), catalase (CAT), and 8-hydroxy-2′-deoxyguanosine (8-OHdG), as well decreased superoxide dismutase (SOD) and glutathione peroxidase-1 (GPx-1) activities, while protective effects were seen for all herbal additives, and better expressed for Centella asiatica and Silybum marianum.