Biochemical and molecular identification of Gram-positive isolates with β-hemolysis activity isolated from the nasal swab of pigs during the human meningitis outbreak in Badung Regency, Bali-Indonesia

Research (Published online: 25-01-2022)

18. Biochemical and molecular identification of Gram-positive isolates with β-hemolysis activity isolated from the nasal swab of pigs during the human meningitis outbreak in Badung Regency, Bali-Indonesia

K. J. Putra Pinatih, I. W. Suardana, I. D. M. Sukrama, I. B. N. Swacita and R. K. Putri

Veterinary World, 15(1): 140-146

ABSTRACT

Background and Aim: The nasal cavity of a pig serves as an entry point and a habitat for the colonization of commensal microbes and pathogenic bacteria. Based on biochemical and serological tests, Streptococcus β-hemolytic Group C was identified as the Gram-positive bacteria, which resulted in the 1994 outbreak and death of thousands of pigs in Bali. Furthermore, this agent is zoonotic and frequently results in the development of meningitis lesions in the infected pig. Recently, a meningitis outbreak in humans was also reported after the consumption of pig-derived foods at Sibang Kaja, Badung-Bali. This study aimed to identify and characterize Gram-positive β-hemolytic organisms collected from nasal swab of pigs from the outbreak area, as well as to compare API Kit and 16S rRNA gene analysis methods.

Materials and Methods: This study commenced with the cultivation of two isolates, Punggul Swab Nasal (PSN) 2 and PSN 19, which were characterized by β-hemolysis activity. These samples were then conventionally and molecularly identified using Kit API 20 Strep and 16S ribosomal RNA (rRNA) gene primers, respectively.

Results: Using the Kit API 20 Strep, both isolates were identified as Enterococcus faecium, which was previously classified as Group D Streptococci. Based on the 16S rRNA gene sequencing, PSN 2 and PSN 19 were molecularly confirmed to have 99 and 98.1% similarities with E. faecium (NR042054), respectively. Furthermore, both isolates share the same clade in the phylogenetic tree analysis.

Conclusion: Using Kit API 20 Strep and 16S rRNA gene analysis, the PSN 2 and PSN 9 Gram-positive isolates with β-hemolysis activity from pig nasal swabs were identified as E. faecium.

Keywords: 16S ribosomal RNA gene, Gram-positive bacteria, Kit API 20 Strep, nasal of pig, phylogenetic tree.

A novel multiplex polymerase chain reaction assay for the genotypic survey of the non-homologous end-joining factor 1 gene associated with Collie eye anomaly in Thailand

Research (Published online: 25-01-2022)

17. A novel multiplex polymerase chain reaction assay for the genotypic survey of the non-homologous end-joining factor 1 gene associated with Collie eye anomaly in Thailand

Chommanad Lerdkrai and Nuch Phungphosop

Veterinary World, 15(1): 132-139

ABSTRACT

Background and Aim: Collie eye anomaly (CEA) is a hereditary and congenital ocular disorder, which affects several dog breeds, including Collies, Collie-related breeds, and other purebreds. An intronic deletion of 7799-bp in the non-homologous end-joining factor 1 (NHEJ1) gene has been identified as the genetic defect causing CEA. This study aimed to investigate the prevalence of CEA based on NHEJ1 genotyping assay in Thailand.

Materials and Methods: We clarified the prevalence of CEA in 224 dogs from five purebred dog breeds using a novel multiplex polymerase chain reaction (PCR)-based technique and confirmed the genotypic status with direct DNA sequencing.

Results: The highest frequency of the mutated NHEJ1 allele was 83.3% for Rough Collies, followed by 7.8% for Border Collies, 5.1% for Australian Shepherds, and 2.8% for Shetland Sheepdogs. The heterozygous mutated NHEJ1 genotype detected for Rough Collies, Border Collies, Australian Shepherds, and Shetland Sheepdogs was 33.3%, 15.6%, 10.3%, and 3.3%, respectively. The homozygous mutated NHEJ1 genotype was detected only in Rough Collies and Shetland Sheepdogs, accounting for 66.7% and 1.1%, respectively. Thai Ridgeback Dogs were not affected by this mutation.

Conclusion: This study describes, for the 1st time, the genotypic survey of the NHEJ1 gene associated with CEA in dogs in Thailand. In addition, we successfully developed a new multiplex PCR assay with high accuracy, reproducibility, and cost-efficiency and validated its usefulness for determining NHEJ1 genotypes.

Keywords: Collie eye anomaly, dogs, multiplex polymerase chain reaction assay, non-homologous end-joining factor 1 genotype.

Cervical anticancer activities of Annona squamosa Linn. leaf isolate

Research (Published online: 24-01-2022)

16. Cervical anticancer activities of Annona squamosa Linn. leaf isolate

Made Dira Swantara, Wiwik Susanah Rita, Made Asmarani Dira and Kadek Karang Agustina

Veterinary World, 15(1): 124-131

ABSTRACT

Background and Aim: Cancer is one of the leading causes of death, the need for new anticancer herbal drugs is becoming more urgent considering the side effects of synthetic drugs. This study aimed to determine the anticancer activity of isolates derived from the methanol extract of Annona squamosa Linn. leaves and to identify the compounds that have an active effect against HeLa cells.

Materials and Methods: The leaf metabolites of A. squamosa L. were extracted using methanol at room temperature (28°C) and were partitioned into n-hexane, chloroform, and n-butanol. The toxicity test of these extracts was conducted using a brine shrimp lethality assay. Furthermore, the most toxic extracts were separated and purified using silica gel column chromatography to yield four isolate fractions: FA, FB, FC, and FD. The most toxic isolates were tested for anticancer against HeLa cells, and their compounds were identified using liquid chromatography-mass spectrometry.

Results: The results showed that the most toxic isolate with an LC50 value of 100.00 ppm had a potency similar to that of an anticancer agent with an IC50 value of 70.9021 ppm. Furthermore, the five compounds identified in this isolate include (6S, 7aR)-6-hydroxy-4,4,7a-trimethyl-6,7-dihydro-5H-1-benzofuran-2-one or loliolide, cocamidopropyl betaine, N-[3- (dimethylamino)propyl]dodecanamide or lauramidopropyl dimethylamine, linolenic acid, and 1-dodecyl-2-azepanone or laurocapram.

Conclusion: It can be concluded that the leaf isolates of A. squamosa Linn. had shown anticancer activities against cervical cancer.

Keywords: Annona squamosa Linn, cervical anticancer activities, HeLa cells.

Comparison of three progesterone quantification methods using blood samples drawn from bitches during the periovulatory phase

Research (Published online: 24-01-2022)

15. Comparison of three progesterone quantification methods using blood samples drawn from bitches during the periovulatory phase

Hassan A. Hussein, Gerhard Schuler, Theresa Conze and Axel Wehrend

Veterinary World, 15(1): 119-123

ABSTRACT

Background and Aim: Measuring blood progesterone (P4) concentration has become an essential diagnostic tool in small animal reproductive medicine. Methods enabling precise and rapid on-site measurements are in high demand, especially for the optimization of breeding management in bitches. This study aimed to compare two commercial on-site methods (Speed™ P4, Virbac [M1] and mini VIDAS®, bioMérieux [M2]) and a well-established radioimmunoassay (RIA), which was used as a reference method.

Materials and Methods: Comparative measurements were performed on 52 blood serum samples collected from 45 clinically healthy bitches of different breeds. The dogs had been presented to determine the estrus cycle stage and predict the time of ovulation. Each sample was divided into three aliquots. In aliquot 1, P4 was measured immediately applying M2. Aliquots 2 and 3 were stored at –20°C until analysis was performed using RIA and M1. The consistency of the three methods was investigated by pairwise linear regression analyses.

Results: In RIA, the P4 concentrations ranged between 1.1 and 25.4 ng/mL. Regression analyses revealed highly significant (p<0.0001) positive correlations between the three methods applied (M1 vs. RIA: R=0.94; M2 vs. RIA: R=0.98; and M1 vs. M2: R=0.91).

Conclusion: The results show that the two commercial on-site methods tested exhibit approximately equal, high consistency with the radioimmunological reference method and can, therefore, be used beneficially in a clinical setting. However, biological interpretation of data must be performed in a method-specific manner.

Keywords: dog, enzyme-linked immunofluorescence, immunochromatography, progesterone, radioimmunoassay.

Seroprevalence of Toxoplasma gondii and associated alterations in hematology and serum biochemistry of one-humped camels (Camelus dromedarius) in Pakistan

Research (Published online: 22-01-2022)

14. Seroprevalence of Toxoplasma gondii and associated alterations in hematology and serum biochemistry of one-humped camels (Camelus dromedarius) in Pakistan

Aamir Shehzad, Awais Masud, Tabassam Fatima, Fraz Munir Khan, Saifur Rehman, Mustofa Helmi Effendi, Lucia Tri Suwanti, Iahtasham Khan, Wiwiek Tyasningsih, Shah Faisal, Zain Ul Abadeen and Samreen Bibi

Veterinary World, 15(1): 110-118

ABSTRACT

Background and Aim: Toxoplasma gondii is an intracellular protozoan that infects humans and animals. This study aimed to estimate the seroprevalence of T. gondii and the associated alterations in hematology and serum biochemistry of one-humped camels (Camelus dromedarius) in Mianwali district, Pakistan.

Materials and Methods: A total of 350 blood samples were obtained from male and female camels of different ages (≤3 years old, 4-6 years old, and ≥7 years old). To validate T. gondii antibodies, the collected samples were subjected to indirect enzyme-linked immunosorbent assay using purified recombinant micronemal protein 3 as an antibody catching antigen.

Results: The prevalence of T. gondii was 50.2% higher in male camels than in female camels (16.5%) (p<0.001). Furthermore, the prevalence of T. gondii in camels was directly proportional to age (p<0.001). It was 63.33% (57/90) in camels of ≥7 years of age, 32.54% in 4-6 years old age group, and 23.08% in ≤3 years old age group. The hematological analysis of infected camels revealed a significant increase in the values of glucocorticoid-remediable aldosteronism, lymphocyte percentage, monocyte percentage (MONO%), corpuscular hemoglobin (MCH), and procalcitonin. Furthermore, substantially higher levels of liver enzymes alanine aminotransferase, aspartate aminotransferase, and the macro-mineral potassium were found in the serum of T. gondii-infected camels.

Conclusion: The seropositivity of T. gondii is directly associated with the age and sex of camels, which may be considered as potential risk factors. Furthermore, T. gondii infection directly impacts the hemato-biochemistry of infected camels.

Keywords: biochemistry, camel, hematology, public health, seroprevalence, Toxoplasma gondii.

The enriched Y-bearing sperm combined with delayed fixed-time artificial insemination for obtaining male Simmental crossbred offspring

Research (Published online: 22-01-2022)

13. The enriched Y-bearing sperm combined with delayed fixed-time artificial insemination for obtaining male Simmental crossbred offspring

Dewa Ketut Meles, Imam Mustofa, Mas'ud Hariadi, Wurlina Wurlina, Suherni Susilowati, Anny Amaliya, Suparto Suparto and Rimayanti Rimayanti

Veterinary World, 15(1): 102-109

ABSTRACT

Background and Aim: The production of male calf beef cattle is an agricultural innovation needed to increase the farm's productivity as a provider of meat sources. This study aimed to determine the sex ratio of the offspring of cows inseminated with Y-bearing sperm enriched by Percoll density gradient centrifugation and swim up, combined with delayed fixed-time artificial insemination (FTAI).

Materials and Methods: Ejaculates of Simmental bulls were divided into four equal portions and grouped as T0 (control, non-sexed semen), T1 and T2 were sexed semen using Percoll density gradient centrifugation three and five levels, respectively, and T3 was sexed semen using swim-up. After the sex was sorted, the semen was diluted in a tris egg yolk extender, packaged in French mini-straws containing 50 million live sperm cells, and frozen. Pre-sexed, post-sexed, and post-thawed spermatozoa were evaluated based on progressive motility, viability, intact plasma membrane, and abnormality. The post-thawed semen of T0 was artificially inseminated to recipient cows at 12 h after onset of estrus (not delayed FTAI). Meanwhile, the delayed FTAI was conducted 18 20 h after onset of estrus using the T0, the best of T1 and T2, and the T3 post-thawed semen.

Results: The Percoll density gradient centrifugation reduced motility, viability, and intact plasma membrane but increased sperm abnormalities. Meanwhile, the swim up process increased motility, viability, and intact plasma membrane of sperm cells but decreased sperm abnormalities. Post-thawed semen decreased motility, viability, and intact plasma membrane of sperm cells but increased sperm abnormalities. The sex ratio of the Simmental crossbred offspring was 96.08% and 100% in T1 and T3, respectively, compared to 48.25% and 67.39% in T0 not delayed and delayed FTAI, respectively.

Conclusion: The Percoll density gradient centrifugation and swim-up methods are prospective for obtaining male offspring.

Keywords: agricultural innovation, farm productivity, motility, pregnancy rate, sperm morphologic abnormality, viability.

Assessment of aflatoxin M1 and B1 in some dairy products with referring to the analytical performances of enzyme-linked immunosorbent assay in comparison to high-performance liquid chromatography

Research (Published online: 22-01-2022)

12. Assessment of aflatoxin M1 and B1 in some dairy products with referring to the analytical performances of enzyme-linked immunosorbent assay in comparison to high-performance liquid chromatography

Raghda Mohamed Esam, Ragaa Shehata Hafez, Nagwa Ibrahim Mohamed Khafaga, Karima Mogahed Fahim and Lamiaa Ibrahim Ahmed

Veterinary World, 15(1): 91-101

ABSTRACT

Background and Aim: Aflatoxin M1 (AFM1) is a major fungal metabolite found in milk coming from aflatoxin B1 (AFB1) contaminated rations and is subsequently present in milk-based products demonstrating a serious public health hazard. This study aimed to investigate the levels of AFM1 and AFB1 in milk and some dairy products consumed widely by infants and children.

Materials and Methods: This study investigated the incidence of AFM1 in 105 samples of processed cheese, Ras cheese, and raw milk (35 of each) retailed in the Egyptian markets. The degree of sensitivity and accuracy was evaluated using the enzyme-linked immunosorbent assay (ELISA) method followed by the estimation of the positive samples using the high-performance liquid chromatography (HPLC) with fluorescence detection. Mold count was determined in the examined samples by investigating AFB1 content using HPLC.

Results: AFM1 was found in all investigated Ras cheese, raw milk, and 82.86% of the processed cheese samples with mean values of 51.05±6.19, 40.27±3.996, and 10.77±1.39 ng/kg, respectively. Moreover, there was statistically no significant difference between AFM1 levels in the core and crust parts of the tested Ras cheese. AFM1 contaminated Ras cheese and raw milk samples were 48.57% and 25.71%, which exceeded the European and Egyptian tolerance levels. Results showed an acceptable correlation between ELISA and HPLC methods with no significant difference (p>0.05). Alternatively, none of the examined samples proved to be contaminated with AFB1 despite the presence of mold with mean counts of 3.79±3.29, 4.39±4.34, and 4.84±4.29 log CFU/g in the examined processed cheese, Ras cheese, and raw milk samples, respectively.

Conclusion: Therefore, it is urgent to regularly inspect the contamination of animal feeds with AFB1 and apply special measures and novel techniques to protect the feed and food from public health hazards.

Keywords: aflatoxin B1, aflatoxin M1, enzyme-linked immunosorbent assay, high-performance liquid chromatography, mold, sensitivity.