Genetic analysis of NS5B gene from bovine viral diarrhea virus-infected cattle in Central and East Java, Indonesia

Research (Published online: 25-07-2019)

27. Genetic analysis of NS5B gene from bovine viral diarrhea virus-infected cattle in Central and East Java, Indonesia

S. H. Irianingsih, H. Wuryastuty, R. Wasito, H. Wibawa, F. S. Tjatur Rasa and B. Poermadjaja

Veterinary World, 12(7): 1108-1115

ABSTRACT

Background and Aim: A previous study divided Indonesian bovine viral diarrhea virus (BVDV)-1 into subgenotypes BVDV-1a to BVDV-1d based on the partial NS5B gene using strain Bega as reference for BVDV-1a. In fact, it is clustered into BVDV-1c with strain Bega-like Australia. BVDV genotyping has been done on isolates from Jakarta, West and Central Java, but East Java isolates have not been genotyped. This study aimed to analyze genetic variability and amino acid residues in the nucleotide-binding pocket of the NS5B gene from infected cattle.

Materials and Methods: Samples were obtained from the Sera Bank originating from active and passive surveillance of cattle that had been tested for BVDV antigen from 2013 to 2017. Detection of the p80 antibody and BVDV genotyping was carried out using ELISA and nested-multiplex-polymerase chain reaction (PCR), respectively. We defined 15 nested PCR products for partial sequencing of NS5B. Those field samples were selected from each location and year using proportional calculation as a representative sample. Homological and phylogenetic analyses of the partial NS5B gene were performed using BLAST and MEGA version 6.

Results: Based on the phylogenetic tree analysis using 360 nucleotides as the partial NS5B gene, Indonesian BVDV-1 isolates from Central and East Java were subdivided to BVDV-1a (n=9), BVDV-1b (n=1), and BVDV-1c (n=5). In the present study, the homology of BVDV subgenotype -1a, -1b, and -1c was compared to the BVDV GenBank data and found 90-93%, 93%, and 92-95% respectively with the average pairwise distance of 0.207. A point mutation was shown at R283K of all BVDV isolates based on the sequence of three amino acid residues R283, R285, and I287 in the nucleotide-binding pocket as a part of the encoded RNA-dependent RNA polymerase.

Conclusion: This study revealed the genetic variability of BVDV infecting cattle in Central Java and East Java, Indonesia, the subtypes BVDV-1a, BVDV-1b, BVDV-1c, and a point mutation at the R283K residue.

Keywords: bovine viral diarrhea virus, NS5B gene, phylogenetic analysis, point mutation, subgenotype.

The concentration of testosterone, pituitary adenylate cyclase-activating polypeptide, and protamine 1 in the serum of male chicken following administration of epididymis and testicular extracts and their combination

Research (Published online: 25-07-2019)

26. The concentration of testosterone, pituitary adenylate cyclase-activating polypeptide, and protamine 1 in the serum of male chicken following administration of epididymis and testicular extracts and their combination

Muslim Akmal, Gholib Gholib, Rinidar Rinidar, Fitriani Fitriani, T. Zahrial Helmi, Sugito Sugito, M. Isa, Nurliana Nurliana, Sri Wahyuni, Dasrul Dasrul and M. Aman Yaman

Veterinary World, 12(7): 1101-1107

ABSTRACT

Background and Aim: Testis and epididymis are male reproductive organs that play an important role in spermatogenesis. These two organs are rich in the content of hormones and other molecules needed in the process of spermatogenesis which affect the quality of the spermatozoa. The objective of this study was to examine the effect of the administration of epididymis and testicular extracts and their combination on testosterone, pituitary adenylate cyclase-activating polypeptide (PACAP), and protamine 1 (PRM1) concentrations in the serum of male chicken.

Materials and Methods: Twenty male chickens (broiler strain Cp707), aged 3 weeks and weighing 800-1000 g, were randomly divided into four different groups including a control group (T0) = injected with 1 ml normal saline and treatment groups: T1 = injected with 1 ml epididymis extract, T2 = injected with 1 ml testicular extract, and T3 = injected with a combination of 1 ml epididymis + 1 ml testicular extract. The experiment was conducted for 13 days and at the end of the study (day 14), the chickens were sacrificed to obtain the serum. Furthermore, the concentrations of testosterone, PACAP, and PRM1 were then measured by using an enzyme-linked immunosorbent assay technique.

Results: The concentrations of PACAP and PRM1 did not show a significant difference between treatment groups (T1, T2, and T3) and control group (T0) (p>0.05). However, the concentration of testosterone showed a significantly higher difference in a group injected with a combination of 1 ml epididymis and 1 ml testicular extracts (T3) compared to the control group (T0) (p<0 .05="" p="">

Conclusion: The administration of epididymis and testicular extracts and their combination did not affect the increase of PACAP and PRM1 concentration. However, a combination of these extracts significantly affects the increase of testosterone concentration in the serum of male chicken.

Keywords: chicken, epididymis and testicular extracts, pituitary adenylate cyclase-activating polypeptide, spermatogenesis, testosterone, protamine 1.

Detection of lumpy skin disease virus in cattle using real-time polymerase chain reaction and serological diagnostic assays in different governorates in Egypt in 2017

Research (Published online: 24-07-2019)

25. Detection of lumpy skin disease virus in cattle using real-time polymerase chain reaction and serological diagnostic assays in different governorates in Egypt in 2017

Gamil Sayed Gamil Zeedan, Ayman Hamid Mahmoud, Abeer Mostafa Abdalhamed, Khaled Abd El-Hamid Abd El-Razik, Manal Hamdy Khafagi and Hala Abdoula Ahmed Abou Zeina

Veterinary World, 12(7): 1093-1100

ABSTRACT

Background and Aim: Lumpy skin disease (LSD), is a highly infectious viral disease of cattle, caused by LSD virus (LSDV) which belongs to the genus Capripoxvirus of family Poxviridae. In the summer of 2017, skin lesions suggestive of LSD were observed in cattle at several governorates in Egypt. This study aimed to detect LSDV in cattle specimens using rapid serological and molecular diagnostic assays.

Materials and Methods: A total of 46 skin biopsies and uncoagulated blood samples were collected from cattle with LSD suggestive clinical signs, as well as 290 coagulated whole blood samples from cattle without skin lesion in different governorates in Egypt during the summer of 2017. Skin biopsies were used for virus isolation from the chorioallantoic membrane of 11-day-old specific pathogen-free embryonated chicken eggs (SPF-ECEs). LSDV was identified using conventional polymerase chain reaction (PCR), real-time PCR (RT-PCR), and fluorescent antibody technique (FAT) with specific hyperimmune serum against LSDV. Cattle sera were examined using indirect FAT (IFAT) and indirect enzyme-linked immunosorbent assay (ELISA).

Results: Skin nodules and sitfast lesions were significant clinical signs observed in all LSD suspect cattle. SPF-ECEs, from which positive isolations were made and it showed characteristic inflammatory and focal white pock lesions. The isolated viruses were identified as LSDV by FAT, conventional gel-based PCR, and RT-PCR. Among the skin biopsies and corresponding blood samples, LSDV-positive samples percentage were 39.13 and 36.95 by RT-PCR, followed 34.78 and 28.26 by conventional PCR and then 32.6 and 26.8 by FAT, respectively. The total positive percentage of LSDV antibody detected in cattle serum samples were 17.93 and 14.48 by indirect ELISA and IFAT.

Conclusion: LSDV was detected and identified in skin biopsies and corresponding blood samples of naturally infected cattle, more LSDV-positive samples were detected by RT-PCR, followed by conventional PCR and then FAT. The indirect ELISA detected more antibody-positive samples than the IFAT from cattle serum samples. The RT-PCR assay is simple, sensitive, rapid, and reliable for the detection of LSDV in blood and skin nodule biopsies of suspected cattle.

Keywords: enzyme-linked immunosorbent assay, indirect fluorescent antibody technique, lumpy skin disease, polymerase chain reaction, Poxviridae, real-time polymerase chain reaction.

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Risk factors associated with cows' lying time, stall and cows' own cleanliness in smallholder dairy farms in Kenya

Research (Published online: 21-07-2019)

24. Risk factors associated with cows' lying time, stall and cows' own cleanliness in smallholder dairy farms in Kenya

E. K. Kathambi, J. A. VanLeeuwen, G. K. Gitau and C. Kamunde

Veterinary World, 12(7): 1085-1092

ABSTRACT

Background and Aim: The welfare of animals kept in livestock production systems has raised concerns around the world. Adult dairy cattle require adequate rest and spend approximately 12 h/day lying down. This cross-sectional study aimed to determine the stall factors and management practices affecting cows' lying time, stall cleanliness, and cows' cleanliness (udder and upper leg), in smallholder dairy cows in Meru County of Kenya.

Materials and Methods: A total of 106 milking cows from 73 farms were assessed for daily lying time and cleanliness. Data loggers were used to record the lying time of cows for 3 days. Stall, udder, and upper leg cleanliness were assessed using a 5-score system: 1 (very clean) to 5 (very dirty). Management information was acquired using a questionnaire that was administered face-to-face to the farmers in their native Kimeru language. Univariable and multivariable linear and logistic regression models were fit to determine factors associated with cows' lying time and dichotomized stall and cows' own cleanliness, respectively.

Results: The mean daily lying time was 10.9±2.2 h, and the mean stall cleanliness score was 2.4±1.0. The mean average cleanliness scores of the udder and upper legs were 1.9±0.7 and 2.5±1.1, respectively. Overall, 35% of the stalls were categorized as dirty (>2.5), whereas 13% and 47% of the cows had udder and leg cleanliness scores >2.5, respectively. From the final multivariable models (p<0 .05="" 1.0="" 5.25="" by="" cleanliness="" conversely="" cows.="" cows="" daily="" decreased="" for="" h="" increased="" lying="" older="" scores="" stall="" than="" time="" versus="" with="" years="" younger="">2.5 and by 1.6 h with poorly positioned neck rails. In an interaction term, addition of new bedding at least once a day without removing stall manure at least once a day decreased the daily lying time of the cows by 1.5 h, whereas failure to add new bedding at least once a day but removing stall manure at least once a day decreased the lying time of the cows by 1.2 h. Farm-level risk factors for stall dirtiness (>2.5) included delayed cleaning of the alley (odds ratio [OR]=6.6, p=0.032), lack of bedding (OR=4.9, p=0.008), and standing idle and/or backward in the stall (OR=10.5, p=0.002). Stalls categorized as dirty (OR=2.9, p=0.041) and lack of bedding (OR=2.7, p=0.065) were cow- and farm-level risk factors for dirtiness of the udder (>2.5), respectively, whereas the stall being dirty (OR=2.3, p=0.043) was the only risk factor (cow level) for dirtiness of the upper legs (>2.5).

Conclusion: It was recommended that farmers should pay attention to the specific factors identified regarding the stall design (e.g., neck rail position) and bedding/manure management that impact the cleanliness of cows and their lying time.

Keywords: dairy cows, Kenya, lying time.

Yersinia enterocolitica: Prevalence, virulence, and antimicrobial resistance from retail and processed meat in Egypt

Research (Published online: 21-07-2019)

23. Yersinia enterocolitica: Prevalence, virulence, and antimicrobial resistance from retail and processed meat in Egypt

Gamal Younis, Mona Mady and Amal Awad

Veterinary World, 12(7): 1078-1084

ABSTRACT

Aim: The objectives of this study were to investigate the prevalence of Yersinia enterocolitica in retail chicken meat, ground and processed beef meat, determine their virulence-associated genes, antimicrobial susceptibility pattern, molecular detection of extended-spectrum β-lactamases, and their capability of biofilm formation in vitro.


Materials and Methods: A total of 210 samples (120 retail chicken meat, 30 ground beef, 30 beef burger, and 30 sausage samples) were collected from different retail chicken outlets and markets located at Mansoura city between December 2016 and April 2017. Meat samples were examined bacteriologically for the existence of Y. enterocolitica; bacterial colonies that displayed positive biochemical properties were subjected to polymerase chain reaction targeting 16 rRNA gene. Y. enterocolitica isolates were tested for their susceptibility to six antimicrobial agents using disk diffusion method. Uniplex PCR was used for screening Y. enterocoliticaisolates for the presence of two virulence chromosome-associated genes (ail and yst), and β-lactamases (blaTEM and blaSHV). The capability of Y. enterocolitica to form biofilms was detected by tube method.

Results:   Thirty Y. enterocolitica isolates (14.29%) were recovered including 19 (15.83%) isolates from chicken meat, 3 (10%) from ground beef, 5 (16.67%) from beef burger, and 3 (10%) from sausage samples. Regarding ail gene, it was detected in 6.67% (2/30), while yst gene detected in 20% (6/30) Y. enterocolitica isolates. About 80%, 70%, 63.33%, and 50% of Y. enterocolitica isolates were sensitive to ciprofloxacin, gentamicin, cefotaxime, and streptomycin, respectively, while 83.33% of Y. enterocolitica isolates were resistant to both ampicillin and cephalothin. Interestingly, 21 (70%) isolates had the capability of biofilms formation in vitro. Among the multidrug-resistant (MDR) strains, a significant difference (p<0 .05="" and="" antimicrobials="" between="" biofilm="" correlated="" formation.="" formation="" found="" genes.="" however="" isolates="" lactam-resistant="" lactam="" mdr="" of="" presence="" resistance="" span="" the="" to="" was="" with="">

Conclusion: The presence of Y. enterocolitica in chicken meat, ground and processed beef meat represents a significant health risk for meat consumers, which reflects the contamination of slaughterhouses and processing operations, therefore, strict hygienic measures should be applied to minimize carcasses contamination.

Keywords: antimicrobial susceptibility, biofilm formation, virulence genes, Yersinia enterocolitica.

Opportunistic pathogenic fungi isolated from feces of feral pigeons in Mafikeng, North West Province of South Africa

Research (Published online: 18-07-2019)

21. Opportunistic pathogenic fungi isolated from feces of feral pigeons in Mafikeng, North West Province of South Africa

Michelo Syakalima, Tsepo Ramatla and Ngoma Lubanza

Veterinary World, 12(7): 1066-1069

ABSTRACT

Background and Aim: Pigeon feces are increasingly being implicated in the spread of bacterial pathogens such as Escherichia coli, Campylobacter, Salmonella, Listeria, and Chlamydia. Fungi are rarely investigated except for Cryptococcus that has emerged as an important pathogen in old people and immunosuppressed patients. This study investigated fungi in pigeon feces collected from Mafikeng, the North West Province of South Africa.

Materials and Methods: Freshly dropped feces were collected and enriched in phosphate-buffered saline overnight at 48°C and then subcultured on Sabouraud's dextrose agar and incubated at 48°C for 2 weeks observing any fungal growth from day 2. The growths were picked up, DNA extracted, and polymerase chain reaction was done using the internal transcribed spacer primers.

Results: Fungi isolated included: Aspergillus (Aspergillus tubingensis), Cryptococcus (Cryptococcus albidus and Cryptococcus randhawai), Fusarium spp., and Rhodotorula (Rhodotorula mucilaginosa and Rhodotorula kratochvilovae). Most of these isolates are known opportunistic pathogens and have been isolated in clinical conditions elsewhere. Other isolates such as Graphium dubautiae, Myrmecridium schulzeri, Naganishia albida, Paecilomyces lilacinus, and Zygopleurage zygospora were not found to be of any human health significance.

Conclusion: We, therefore, concluded that the presence of these opportunistic pathogens is a significant human health risk, especially in the face of the HIV/AIDS pandemic that results in immunosuppression.

Keywords: chain reaction, fungi, opportunistic pathogens, pigeon feces, polymerase.

Weissella, a novel lactic acid bacteria isolated from wild Sumatran orangutans (Pongo abelii)

Research (Published online: 18-07-2019)

20. Weissella, a novel lactic acid bacteria isolated from wild Sumatran orangutans (Pongo abelii)

Safika Safika, Wardinal Wardinal, Yulia Sari Ismail, Khairun Nisa and Wenny Novita Sari

Veterinary World, 12(7): 1060-1065

ABSTRACT

Aim: This study aimed to isolate and identify lactic acid bacteria (LAB) in wild Sumatran orangutans to provide more information about LAB diversity derived from Sumatran orangutan feces.

Materials and Methods: Fecal sampling from three female orangutans, around 35 years old, was carried out in the wild forest areas at the research station of Suaq Belimbing Gunung Leuser National Park located in the South Aceh district. Orangutan fecal samples were taken in the morning when the orangutans first defecated. The orangutans were above the tree, which is approximately 12-15 m from the ground where feces were found.

Results: Fermentation testing using the API 50 CHL Kit showed that OUL4 isolates were identified as Lactococcus lactis ssp. lactis with an identity value of 73.5%. Homology analysis demonstrated that the OUL4 isolates have 93% similarity to Weissella cibaria, and phylogenetic trees constructed using Mega 7.0 also showed that OUL4 isolates are related to W. cibaria.

Conclusion: These results show that there is a difference in identification between biochemical testing with API kits and molecular analyses on LAB isolates from wild Sumatran orangutans. Based on 16S rRNA gene homology, the OUL4 LAB isolates from wild Sumatran orangutans have 93% homology to W. cibaria.

Keywords: 16S rRNA, API 50 CHL, lactic acid bacteria, orangutan, Pongo abelii, Weissella.