Biological Factors Associated with Infectious Diarrhea in Calves

Calf diarrhea is a major cause of economic loss in the cattle industry. Identifying biomarkers associated with the pathological conditions may provide beneficial guidelines for detecting disease progression and monitoring effectiveness of therapeutic agents in calves suffering from diarrhea. The objective of this research was to find out biological factors associated with calf diarrhea, based on clinical findings and the presence of enteric pathogens. Fecal and blood samples were obtained from 73 non-diarrheal and 42 diarrheal calves. The presence of enteric pathogens, hemato-chemical parameters, acute phase proteins, and pro-inflammatory cytokines were investigated in the samples. According to the presence of diarrhea and pathogens, the calves were classified into healthy (n=35), subclinical (non-diarrheal but pathogen-positive, n=36), infectious diarrheal (diarrheal and pathogen-positive, n=39), and unknown diarrheal groups (diarrheal but pathogen-negative, n=3). The presence of bovine coronavirus, bovine rotavirus group A, and Cryptosporidium spp. were significantly associated with the development of calf diarrhea (P<0.05). The infectious diarrheal group showed increases in monocyte percentages and blood urea nitrogen level and the decreases in glucose, potassium, and phosphorus levels (P<0.05). Moreover, inflammatory proteins such as haptoglobin, fibrinogen, interleukin-6, and tumor necrosis factor-alpha were elevated in the infectious diarrheal group compared to the healthy group (P<0.05). The current study identified altered blood biological factors in calves with infectious diarrhea. The results suggested that these factors may be useful targets for monitoring animal health in calf diarrheal disease, especially infectious diarrhea.

Dose Related Analgesic, Motor and Reinforcing Effects of Nalbuphine in Rats

Nalbuphine, a semi-synthetic opioid drug, is a kappa (κ) agonist/ mu (μ) partial agonist. It is clinically used for moderate to severe pain. It produces the analgesic effect largely by binding to kappa opioid receptors. The present study was designed to investigate locomotor sensitization as well reinforcing effects of different doses (5, 10 and 20 mg/kg) of nalbuphine in rats. Potential analgesic and hyperalgesic effects after single and repeated administration respectively were also monitored. Reinforcing effects were monitored in a conditioned place preference (CPP) paradigm and associated changes in motor activity were monitored during a drug conditioning phase. The hot plate test was used to monitor nociceptive response. The present study showed that low (5 mg/kg) and high (20 mg/kg) doses of nalbuphine were reinforcing, while the moderate dose (10 mg/kg) had no reinforcing effect in the CPP paradigm. All doses were analgesic after the first administration and on repeated administration hyperalgesia did not develop to any dose. Analgesic effects still occurred at moderate doses of nalbuphine. Sensitization-like effects were produced following moderate and high doses of nalbuphine. These findings suggested that a moderate dose of nalbuphine did not produce reinforcing effects and hyperalgesia so this dose can be used safely for treating pain.

Evaluation of the Inhibitory Effects of Coumermycin A1 on the Growth of Theileria and Babesia Parasites in vitro and in vivo

Coumermycin A1, a coumarin antibiotic, has anticancer, antibacterial, antiviral, and antimalarial activities. We aimed to evaluate the anti-thielerial and anti-babesial activity of coumermycin A1 in mice in vivo. Coumermycin A1 efficacy was determined by the transcription of DNA gyrase, a type II DNA topoisomerase using reverse transcriptase-polymerase chain reaction (RT-PCR) transcription. Coumermycin A1 significantly inhibited the development of preliminary parasitemia (1%). Theileria equi and the Babesia species B. bigemina, B. bovis, and B. caballi were observed with IC50 values of 80, 70, 57, and 65 nM, respectively. Their development was remarkably inhibited at observed concentrations of 10, 25, 50, and 100µM for the studied organisms T. equi, and the Babesia species B. caballi, B. bovis and B. bigemina, respectively. In the subsequent viability test, parasite re-growth was suppressed at 100µM for B. bigemina and B. bovis and at 50 µM for B. caballi and T. equi. Coumermycin A1 Treatment of B. bovis cultures with Coumermycin A1 completely suppressed the transcription of the DNA gyrase subunits B and A genes. In BALB/c mice, the development of Babesia microti was inhibited by 70.73% using 5 mg/kg of Coumermycin A1.

Orientin Attenuates Cisplatin-Induced Renal Toxicity by Reducing Oxidative Stress and Inflammation

Cisplatin (CP), an effective chemotherapeutic drug, has been widely used to treat the several types of tumors. Orientin (ORI) is a flavonoid that shows versatile therapeutic activities. The current research was planned to observe the protective role ORI on CP induced renal injury in rats. Twenty-four male rats were divided into four groups equally and termed as control, CP (10 mg/kg), CP (10 mg/kg) + ORI (40 mg/kg) and ORI (40 mg/kg). After seven days trial, rats were dissected and different parameters were analyzed. Results indicated that the CP administration significantly reduced the activities of catalase, peroxidase, glutathione reductase and glutathione content whereas it increased the level of hydrogen peroxide and TBARS (thiobarbituric acid reactive substances). CP increased the creatinine and urea levels while decreased the creatinine clearance. Moreover, CP significantly increased the inflammatory markers, including nuclear factor kappa-B, tumor necrosis factor-α, Interleukin-6, Interleukin-1β levels, cyclooxygenase-2 activity and histopathological damages. However, co-administration of ORI displayed curative effects against CP-induced renal toxicity and recovered all parameters by bringing them to a normal level. These results revealed that the ORI is a potential bioflavonoid that can potentially counter the CP-induced renal damage.

Antimicrobial Effect of Essential Oils on Multidrug-Resistant Salmonella typhimurium in Chicken Fillets

Chicken meat, which could be a healthy and nutritionally food, is regrettably incriminated as a source of Salmonella typhimurium which has a great ability to cause human salmonellosis. This study aimed to assess the prevalence of multidrug-resistant S. typhimurium in chicken meat and the effects of essential oils on its viability. A total of 300 chicken meat and its products samples were streaked on XLD agar plates, which was followed by identification of the isolates based on biochemical and serological tests. Ten isolates were serotyped as S. typhimurium then assayed for susceptibility to 14 antimicrobials by the single diffusion method. Eight isolates (80%) showed multiple antimicrobial resistance (MAR) for 3 or more antimicrobials with MAR index of 0.4857 in average. Serotyped S. typhimurium strain with the highest antimicrobial resistance, confirmed by 16s RNA sequencing, was selected for studying the effects of thyme, oregano, and lemon essential oils with concentrations of 0.5, and 1% on its viability after inoculating into chicken fillets by intensity of 3.0×106 and on sensory traits of chicken fillets on 2nd, 4th, 6th, and 8th days of inoculation during cold storage (4ºC). All results showed a significant reduction of S. typhimurium counts with highest inhibition obtained using 1% lemon essential oil. The sensory properties of treated chicken fillets were improved by all used essential oils, compared to the control samples after 6th day, and 8th day of the storage period. The samples treated with 0.5, and 1% lemon essential oil revealed the highest improvement of sensory attributes. This study proved that the majority of S. typhimurium existing in chicken meat are Multidrug-resistant and have no negative effect on sensory traits, hence, posing a public health hazard. Natural essential oils have, also, great antimicrobial effect on S. typhimurium, thus it could replace chemical antimicrobials.

Digit Bones (Acropodium) of Gazella (Gazella subgutturosa); Three-Dimensional Modelling and Morphometry

The present study was conducted to identify morphometric values and to reveal differences between sexes through three-dimensional modeling of digit bones (ossa phalanges) of gazella by using computed tomography images. A total of 14 (7 female, 7 male) adult gazella (Gazella subgutturosa) cadavers were used in the study. The images were extracted by scanning acropodium bones of gazella at 64-detector Multi Detector Computed Tomography (MDCT) in cross section thickness of 0.625mm. Using MIMICS 20.1 program, three-dimensional models of these images were prepared and morphometric measurements were taken. In GLpe and Bd measurements of Phalanx proximalis, a statistically significant difference was established between females and males in terms of the left forelimb internal, the right forelimb internal, the right forelimb external, and the right hindlimb external bones (P<0.05). There was a statistically significant difference between females and males in terms of volume of phalanx proximalis and surface area of the right forelimb internal phalanx proximalis (P<0.001). In GLpe measurements, a statistically significant difference was determined between females and males in terms of the left forelimb internal, the right forelimb internal, the right hindlimb internal, the left forelimb external, the right forelimb external, and the right hindlimb external phalanx media (P<0.05). A statistically significant difference was determined between females and males in terms of the data related to volume and surface areas of the left hindlimb internal the right hindlimb internal, the left hindlimb external, and the right hindlimb external phalanx distalis (P<0.05).

Construction of a Triple Gene-Deleted Live Vaccine Candidate against Pseudorabies Virus Using CRISPR/Cas9 and Cre/LoxP System-Based Strategy

The immune protective effects of pseudorabies virus (PRV) vaccines in the market have gradually reduced and they have failed to provide complete protection against the new PRV variant. In this study, a triple gene-deleted live PRV strain–rZDΔTK-gE-gI was successfully constructed by simultaneously knocking out the three major virulence genes (gE/gI and TK) with CRISPR/Cas9 and Cre/LoxP gene editing system and low melting point agarose purification method. After challenge with the virulent PRV variant, all the 3-week-old piglets vaccinated with the rZDΔTK-gE-gI PRV vaccine candidate survived without any clinical symptoms, whereas all the unvaccinated piglets exhibited PRV respiratory and neurological signs with 100% mortality rate within 7 days post infection. High levels of anti-gB antibodies were induced in the vaccinated piglets after vaccination with the rZDΔTK-gE-gI vaccine candidate, which elicited a better immune protective effect than the classical strain Bartha-K61. Therefore, the triple gene-deleted live PRV vaccine candidate is expected to control the current outbreak of pseudorabies caused by the PRV variants.

Frequency and Antimicrobial Susceptibility of Methicillin and Vancomycin-Resistant Staphylococcus aureus from Bovine Milk

The increase in resistance pattern of Staphylococcus aureus (S. aureus) has been an emerging threat in therapeutic areas of the dairy industry throughout the globe. The current study was conducted in bovines of district Faisalabad, Pakistan to investigate the phenotypic prevalence of methicillin-resistant (MRSA) and vancomycin-resistant S. aureus (VRSA) in milk samples positive for subclinical mastitis. The study further aimed to assess the associated risk factors and antimicrobial susceptibility pattern against MRSA and VRSA isolates. A total of 385 milk samples (n=193 cattle; n=192 buffalo) collected and screened for subclinical mastitis by surf field mastitis test (SFMT) were further subjected to standard microbiological techniques for the isolation of S. aureus. The positive isolates of S. aureus were phenotypically evaluated for MRSA and VRSA by the disc diffusion method. The study results revealed that out of 385 milk samples, 45.97% (177/385) samples were found positive for subclinical mastitis on SFMT while 37.14% (143/385) samples were confirmed for the presence of S. aureus. Out of these S. aureus isolates, MRSA and VRSA were confirmed in 17.48% (25/143) and 12.58% (18/143) samples respectively. The in-vitro trials of various antibiotics for MRSA and VRSA isolates showed 100% resistance towards Cefoxitin followed by 50% towards Gentamicin, Tylosin, and Trimethoprim + Sulfamethoxazole then 25% to Oxytetracycline, and Fusidic acid while ciprofloxacin, moxifloxacin, and linezolid were found sensitive against study isolates. The public health importance of S. aureus and emerging resistance against antibiotics like methicillin and vancomycin demands regular monitoring of effective use of antimicrobial agents against the isolates of VRSA and MRSA.

An Economical Non-Antibiotic Alternative to Antibiotic Therapy for Subclinical Mastitis in Cows

The economic importance of mastitis and antibiotic resistance is dictating to search non-antibiotic alternatives for the therapy. Trisodium citrate (TSC) being buffer system of the glandular tissue and, vitamin C (Vit. C), zinc and copper being important ingredients required for functioning of immune system fancy chances for a suitable alternative mastitis therapy. The current study was planned to evaluate therapeutic efficacy and cost effectiveness of these ingredients in subclinical mastitis. For this purpose, 40 sub-clinically mastitis cows were randomly divided into 2 equal groups. Group T1 was treated orally with TSC, Vit. C, ZnSo4 and CuSo4, while group T2 was treated with standard antibiotic therapy. Milk pH significantly (P<0.05) differed between the two treatments till day 7th post-initiation of treatment when T1 restored the pH values within normal range earlier than T2. A non-significant (P>0.05) difference was observed in milk pH, fat, lactose, proteins, TS, SNFs, somatic cell counts and restoration of milk yield between the two treatments indicting comparable efficacy. A statistically significant (P<0.05) difference was observed in serum Cu and Zn levels indicating that the supplementation of Cu and Zn led to higher serum values in animals of T1. The use of non-antibiotic oral formulations as mastitis therapy resulted in a net profit of Rs. 457/animal/day. The oral non-antibiotic antibacterial formulation is a therapeutically and economically suitable alternative to rational antibiotic-based therapy to treat subclinical mastitis in dairy cows.

Genetic Characterization of Equine Herpesvirus 1 from Clinical Cases and Asymptomatic Horses in Serbia and Bosnia and Herzegovina

Equine herpesvirus 1 (EHV-1) causes considerable economic loss to the equine industry and is spread among susceptible animals during the cycles of latency and reactivation, causing rhinopneumonitis, abortion, and neurological disease. Nucleotide polymorphisms within ORF30 and ORF68 sequences of the viral genome are associated with strain neuropathogenicity and geographical origin. A total of 142 tissue and nasal swab samples from apparently healthy unvaccinated horses were examined to ascertain EHV-1 distribution, diversity, and clinical significance considering the results of virus isolation, sequence analysis, and anamnestic data. The ORF30 and ORF68 molecular study of these circulating strains and archival isolates from abortion storms aimed to contribute to the perception of strain pathogenicity and origin. EHV-1 was detected by PCR and virus isolation in 81 and 45.1% of the analyzed samples, respectively, and 82.1% of the representative samples were neuropathogenic strains. The ORF68-based grouping was restricted by the pronounced polymorphism of Balkan EHV-1 strains, and only two isolates were assigned to group 4. The cases of abortion were caused by neuropathogenic strains that also circulate within the horse population with no documented outbreaks of disease. It was evident that strain virulence is not solely accountable for the development of clinical symptoms in affected animals. Neural tissue is significant for virus latency and reactivation, considering the number of EHV-1 isolates from apparently healthy stressed horses. Special care must be taken when accommodating together immunologically naive and latently infected horses since asymptomatic carriers silently shed EHV-1.