Development of a multiplex polymerase chain reaction technique for detection and discrimination of Eimeria spp. in cattle in Indonesia

Research (Published online: 18-04-2022)

22. Development of a multiplex polymerase chain reaction technique for detection and discrimination of Eimeria spp. in cattle in Indonesia

Fitrine Ekawasti, Raden Wisnu Nurcahyo, Mukh Fajar Nashrulloh, Dwi Priyowidodo and Joko Prastowo

Veterinary World, 15(4): 975-980

ABSTRACT

Background and Aim: Bovine eimeriosis is a disease caused by apicomplexan parasites of the genus Eimeria. It is one of the most important and widespread bovine illnesses in the world. Some of the identified species of bovine eimeriosis have morphologically similar oocysts that are difficult to differentiate. For the identification of particular Eimeria spp., diagnostic laboratories are increasingly turning to DNA-based technology. This study aims to develop a multiplex polymerase chain reaction (mPCR) technique based on the internal transcribed spacer-1 (ITS-1) gene for the simultaneous identification of pathogenic Eimeria spp. in cattle from Sulawesi Island, Indonesia.

Materials and Methods: Genomic DNA was extracted by the DNAzol reagent from the purified Eimeria oocysts. Species-specific primers targeting the ITS-1 region were used to amplify the distinct Eimeria spp.

Results: Using PCR ITS-1, this study showed that 36 of 120 fecal samples (30%) were infected by Eimeria spp. The multiplex PCR assay allowed for the simultaneous identification of six major Eimeria spp. in a single-tube reaction. The proportion of mixed Eimeria spp. infections was 100% (36/36). The maximum number of Eimeria spp. was five, and the minimum number was two.

Conclusion: Identification of six pathogenic Eimeria spp. in cattle was successfully carried out by nested multiplex PCR using ITS-1 gene. In the future, a procedure to detect pathogenic Eimeria spp. in one tube reaction will offer economical and save diagnostic time.

Keywords: bovine, diagnostic, eimeriosis, multiplex, nested, Sulawesi island.

The first study on confirmation and risk factors of acute and chronic canine distemper in stray dogs in Wasit Province, Iraq, using enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction

Research (Published online: 18-04-2022)

21. The first study on confirmation and risk factors of acute and chronic canine distemper in stray dogs in Wasit Province, Iraq, using enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction

Hadeel Asim Mohammad, Eva Aisser Ajaj, and Hasanain A. J. Gharban

Veterinary World, 15(4): 968-974

ABSTRACT

Background and Aim: In Iraq, stray dogs represent a critical population of free-roaming animals, which probably play a role in the transmission of different infections to other animals. Canine distemper is one of the most growing viral threats to carnivores in many countries worldwide, including Iraq. Therefore, this study was aimed to diagnose the disease using serological and molecular assay and the role of risk factors in the spreading infection.

Materials and Methods: In all, 158 venous blood samples were collected randomly from stray dogs in rural and sub-urban areas of Iraq from May 2019 to December 2020. The samples were examined serologically using two enzyme-linked immunosorbent assay (ELISA) kits, immunoglobulin G (IgG) and immunoglobulin M (IgM), and molecularly by reverse transcription-polymerase chain reaction (RT-PCR) to detect and confirm chronic and acute infections. To determine the association between infection and various risk factors, the study animals were divided according to their locations, sexes, and ages. The age groups were ≤8 months (puppy), 1-3 years (young), and ≥3 years (old/mature).

Results: ELISA result shows that 6.96% and 19.62% of dogs were seropositive for acute and chronic distemper, respectively. The titer of chronic infections (0.421±0.027) was significantly higher (p≤0.025) than that of acute canine distemper (0.337±0.016). On RT-PCR, 8.86% of dogs were found positive for distemper. Using RT-PCR as the gold standard, the sensitivity and specificity of the IgM ELISA kit were 75% and 98.63%, respectively, whereas the positive and negative predictivity were 81.82% and 97.96%, respectively. A significant variation (p<0.05) was observed in the distribution of positive findings among the different epidemiological risk factors. Compared with rural areas, positivity was significantly higher (p<0.05) in sub-urban areas on IgM (26.92%) and IgG (64.15%) ELISA and RT-PCR (34.62%). On IgM ELISA and RT-PCR, no significant differences (p>0.05) were found among the three age groups; however, positivity was significantly higher (p≤0.048) in the ≥3 years group (22.73%) on IgG ELISA. Furthermore, only IgG ELISA showed a significantly higher (p≤0.032) positivity rate in female dogs (25.23%) than in male dogs (7.84%).

Conclusion: To the best of our knowledge, this is the first Iraqi study to demonstrate acute and chronic canine distemper in stray dogs, suggesting that the prolonged shedding of virus from positive dogs is a critical point in the epidemiology of the disease. Additional studies in dogs or other carnivores are required to establish baseline data on the prevalence of the disease in Iraq.

Keywords: epidemiology, Iraq, molecular, Morbillivirus, serology.

Spontaneous expression of the gene of KI67 and P53 in cynomolgus monkeys infected with papillomavirus

Research (Published online: 17-04-2022)

20. Spontaneous expression of the gene of KI67 and P53 in cynomolgus monkeys infected with papillomavirus

Huda S. Darusman, Sela S. Mariya, Isti K. Sari, Maulida A. Nisa, Kartika Sari, Silmi Mariya, Apon Zaenal Mustopa and Uus Saepuloh

Veterinary World, 15(4): 962-967

ABSTRACT

Background and Aim: Cynomolgus monkeys (Macaca fascicularis) develop spontaneous infection of Papillomavirus (PV); thus, potentially beneficial for modeling human PV (HPV) infection study. Contrary to human origin, infection in cynomolgus monkeys does not always show evident clinical symptoms of cervical cancer. The absence of cervical cancer clinical symptoms leads us to investigate the molecular mechanism of the HPV infection in cynomolgus monkeys. This study aimed to investigate the messenger ribonucleic acid (mRNA) expression levels of KI67 and P53 genes, majorly known as biomarker oncogenesis of PV infection.

Materials and Methods: The polymerase chain reaction (PCR) technique was used with MY11/MY09 primer to screen PV in cynomolgus monkey, further grouped as positive-PV and negative-PV infection groups. Real-time quantitative PCR was also applied to quantify the mRNA expression levels of KI67 and P53 genes in animals.

Results: Increased expression of mRNA level of KI67 genes was significantly higher in Positive- PV group than negative-PV group. In contrast, the P53 mRNA expression level increased markedly higher in the negative-PV group than in the positive-PV group.

Conclusion: Our study describes the potential of cynomolgus monkeys as a spontaneous oncogenesis model of PV infection-type. However, we used a limited number of cancer genetic markers. So, further study of other genetic markers is required to prove that cervical cancer could be developed naturally in cynomolgus monkeys.

Keywords: animal model, biomarker genes, cervical cancer, nonhuman primate.

Identification of the molecular characteristics of Bacillus anthracis (1982-2020) isolates in East Indonesia using multilocus variable-number tandem repeat analysis

Research (Published online: 16-04-2022)

19. Identification of the molecular characteristics of Bacillus anthracis (1982-2020) isolates in East Indonesia using multilocus variable-number tandem repeat analysis

D. W. Yudianingtyas, B. Sumiarto, H. Susetya, Mo Salman, T. F. Djatmikowati, Haeriah Haeriah, Abdul Rahman and R. Mangidi

Veterinary World, 15(4): 953-961

ABSTRACT

Background and Aim: Anthrax is one of the endemic strategic diseases in East Indonesia, particularly in the provinces of South Sulawesi, West Sulawesi, Gorontalo, East Nusa Tenggara, and West Nusa Tenggara. Anthrax is an important disease due to its zoonotic and economic impact on the livestock industry. This study aimed to identify the molecular characteristics of Bacillus anthracis in East Indonesia using multilocus variable-number tandem repeat (VNTR) analysis (MLVA).

Materials and Methods: Isolates were obtained from an investigation of anthrax outbreaks in five provinces of East Indonesia from 1982 to 2020. Conventional polymerase chain reaction for B. anthracis was used to identify MLVA-8. Deoxyribonucleic acid sequencing analysis was based on MLVA-8 primers for VNTR identification of the phylogenetic relationship among 24 isolates of B. anthracis obtained from 17 distinct districts/cities in East Indonesia. Tandem Repeats Finder was used for VNTR identification, and Molecular Evolutionary Genetics Analysis X was used to construct phylogenetic analysis.

Results: In this study, 24 isolates were classified as genotype or lineage A. There were four subgroups of B. anthracis circulating in East Indonesia based on eight molecular marker loci sequence results.

Conclusion: The findings of this study show that MLVA-8 typing might be useful as a subtyping tool for the epidemiological investigation of identical genotypes and low genetic diversity of B. anthracis. No other lineage of B. anthracis was circulating in East Indonesia. Other molecular methods are needed, such as extended MLVA, whole-genome sequencing, and canonical single-nucleotide polymorphism, for a more precise study of B. anthracis genetic diversity.

Keywords: Bacillus anthracis, Indonesia, multilocus variable-number tandem-repeat analysis-8.

Age features and reference intervals for the concentrations of some essential and toxic elements in laying hens

Research (Published online: 15-04-2022)

18. Age features and reference intervals for the concentrations of some essential and toxic elements in laying hens

Svyatoslav Lebedev, Oleg Zavyalov and Aleksey Frolov

Veterinary World, 15(4): 943-952

ABSTRACT

Background and Aim: Micronutrient imbalances pose a severe threat to the health and productivity of livestock and poultry. In this regard, a further stage in feeding science development will control and optimize the intake of mineral substances, including determining the elemental composition in some biosubstrates. One of these biosubstrates can be a feather. However, the amount of available information on the content of trace elements in laying hens is limited, complicating the laboratory data interpretation. Therefore, this study established reference intervals for the concentrations of the main essential and toxic elements in laying hens in different periods of ontogenesis.

Materials and Methods: The study was conducted on clinically healthy Hisex Brown laying hens at the age of 10 (n=150), 30 (n=150), 120 (n=150), 150 (n=150), and 210 (n=150) days. All examined birds were born and raised on the territory of the South Ural biogeochemical province of Russia. The sampling of feathers was carried out by plucking the flight feathers of the wing. Inductively coupled plasma dynamic reaction cell mass spectrometry determined the elemental composition of the feather according to 25 parameters.

Results: The results showed that at the minimum age (10 days), the highest concentrations of chemical elements were observed in laying hens. Subsequently, as they grew older, in the period from the 30th to the 120th day, there was a significant decrease in these indicators. Later, from the 120th to the 150th day, a statistically significant increase in concentrations was replaced. Little growth and relative concentration stability were observed in the last part of the experiment (150-210 days). Chemical element concentrations in feathers were computed in reference ranges for each age group.

Conclusion: The calculated ranges of chemical element concentrations in laying hens can be used to compile norms for their content in the body; however, it is worth noting that these ranges can vary depending on the biogeochemical province of breeding and the bird's age.

Keywords: age, essential and toxic elements, feathers, laying hens, reference intervals.

Hydrolyzation of snail (Achatina fulica) meat with rice water as novel probiotic supplements for animal feed

Research (Published online: 15-04-2022)

17. Hydrolyzation of snail (Achatina fulica) meat with rice water as novel probiotic supplements for animal feed

Ujang Suryadi, Rosa Tri Hertamawati and Shokhirul Imam

Veterinary World, 15(4): 937-942

ABSTRACT

Background and Aim: Snail meat and digestive tract hydrolyzate fermented with a consortium of preserved rice water microorganisms could serve as new sources of probiotics. Microorganisms from the examined feed supplement were isolated, identified, and characterized for resistance at low pH and with bile salts. The study aimed to determine the potential hydrolysate of the snail meat and digestive tract as a novel probiotic supplement for animal feed at various pH values and Oxgall.

Materials and Methods: The submerged fermentation method was conducted for 21 days to examine the novel probiotic that originated from snail microorganisms in the hydrolyzed liquid fermented by finely ground snail meat and the digestive tract. The microorganisms in the hydrolyzate were isolated by a spread plate method, while the potential of the probiotic hydrolyzate was tested for resistance to pH values of 2, 2.5, 3, and 4, as well as resistance to bile salts at Oxgall concentrations of 0.2%, 0.3%, 0.5%, 1%, and 2%.

Results: The hydrolyzate profile of snail meat and digestive tract contained five isolates of lactic acid bacteria that could serve as potential probiotics.

Conclusion: The application of fermentation technology using a consortium of preserved rice water microorganisms can convert snail meat and the digestive tract into novel probiotic products that could be utilized in feed supplements.

Keywords: bile, pH, probiotics, snails, supplements.

Hepatoprotection of Cinnamomum burmannii ethanolic extract against high-fat and cholesterol diet in Sprague–Dawley rats (Rattus norvegicus)

Research (Published online: 15-04-2022)

16. Hepatoprotection of Cinnamomum burmannii ethanolic extract against high-fat and cholesterol diet in Sprague–Dawley rats (Rattus norvegicus)

Retno Susilowati, Abdul Malik Setiawan, Afida Fatimatuz Zahroh, Zadani Nabila Ashari, Alifatul Iffiyana, Ricky Hertanto, Muhammad Basyarudin, Isnaeni Hartiningsih and Mahrus Ismail

Veterinary World, 15(4): 930-936

ABSTRACT

Background and Aim: The pathogenesis of non-alcoholic steatohepatitis involves non-alcoholic fatty liver, oxidative stress, inflammation, and fibrosis. Although the long-term use of cinnamon bark in larger doses can negatively affect good health, proper use of its extracts effectively and efficiently improves health. Therefore, this study aimed to determine the minimal dose of Cinnamomum Burmannii extract through its activity in inhibiting oxidative stress in rats' livers treated with a high-fat and cholesterol diet (HFCD).

Materials and Methods: Forty-two Sprague–Dawley rats (Rattus norvegicus), weighing 200-250 g body weight (BW), were divided into seven treatment groups with six replications: Normal, HFCD, atorvastatin, quercetin, and C. burmannii ethanol extract group, after which they were administered different dosages (i.e., 100, 200, and 300 mg/kg BW). Except for the normal group, rats were concomitantly administered HFCD with each treatment for 21 days. Then, their malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity were assessed using colorimetry. However, their steatosis levels were determined based on histological preparations with hematoxylin-eosin staining.

Results: Duncan's multiple range test (DMRT) results indicated that all treatments had a significantly lower MDA than HFCD and normal rats (α=0.01). DMRT results also showed that treating with the C. burmannii ethanol extract at all dosages resulted in a significantly higher SOD activity level in HFCD rats than those treated with quercetin and atorvastatin (α=0.01). Furthermore, results showed that treatment with C. burmannii extracts at a dosage of 300 mg/kg BW incredibly maintained SOD activity as effective as quercetin, atorvastatin, and normal rats. Besides, while steatohepatitis levels of C. burmannii ethanol extract at dosages of 200 and 300 mg/kg BW commensurated with normal rats, steatohepatitis levels were significantly lower than those administered other concentrations or treatments (α=0.05).

Conclusion: Ethanolic C. burmannii extracts protected the liver by regulating oxidative stress. Therefore, a 200 mg/kg BW dose is proposed as the minimal hepatoprotection dose to prevent fatty liver formation.

Keywords: Cinnamomum burmannii, hepatoprotection, malondialdehyde, steatohepatitis, superoxide dismutase.