April H. Wardhana
Balai Besar Penelitian Veteriner Bogor Jl R.E. Martadinata No. 30, Bogor

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Molecular identification technique of Trypanosoma evansi by Multiplex Polymerase Chain Reaction Sawitri, Dyah H.; Wardhana, April H.; Wibowo, H.; Sadikin, M.; Ekawasti, Fitrine
Jurnal Ilmu Ternak dan Veteriner Vol 20, No 4 (2015): DECEMBER 2015
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (382.422 KB) | DOI: 10.14334/jitv.v20i4.1248

Abstract

Trypanosoma evansi is a Hemoflagella parasite that infects cattle and is known as the agents of Surra. Several other trypanosome species infects mammals: T. equiperdum, T. b. rhodesiense, T. b. gambiense, T. vivax, T. congolense, T.theileri. Some of these species is quite difficult to be distinguished morphologically with T. evansi through conventional techniques (thin blood smear). Molecular technique by polymerase chain reaction (PCR) is reported to have the ability to identify, characterize and diagnose trypanosomes accurately. However, a single PCR used is relatively expensive because it takes at least two or more pairs of primers to determine T. evansi. The purpose of this study is to develop T. evansi species identification techniques by multiplex PCR/mPCR (the three pairs of primer in one reaction) that takes the relatively fast and inexpensive. A total of 31 isolates T.evansi were obtained from Bblitvet Culture Collection (BCC) and the Department of Parasitology BBLitvet used in this study. Isolates represent isolates from endemic areas and Surra outbrake isolated from 1988-2014. DNA extraction performed on each sample, including Bang 87 isolates which has been purified as a positive control. Primers used are specific for T. evansi, the ITS-1, Ro Tat 1.2 VSG and ESAG 6/7. Before running mPCR, each primer is optimized by using a single PCR. The results showed that the three primers can be combined in a single reaction with mPCR technique and amplify each DNA fragment target perfectly, so identified 31 isolates as T. evansi. This technique can be applied in the field with a lower cost and faster time.Key Words: Trypanosoma evansi, Identification, Multiplex PCR
Evaluation of mating behaviour and mating compatibility methods for the Old World screwworm fly, Chrysomya bezziana. Wardhana, April H.; MM, Cameron; S, Muharsini; MJR, Hall
Indonesian Journal of Animal and Veterinary Sciences Vol 18, No 4 (2013)
Publisher : Indonesian Animal Sciences Society

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (237.362 KB) | DOI: 10.14334/jitv.v18i4.333

Abstract

The effectiveness of the Sterile Insect Technique program (SIT) to eradicate pest insects relies on the success of mating competitiveness between irradiated male flies and wild type males for the wild type females. It has been successfully applied for the New World screwworm fly (NWSF), Cochliomyia hominivorax but remains unproven for the Old World screwworm fly (OWSF), Chrysomya bezziana. The aim of the study was to develop methods for investigating mating behaviour and mating compatibility of C. bezziana under laboratory conditions. Two methods were used for studying mating: individual mating (method 1) and group mating (method 2). The flies used in this study were 5-7 days old. Twenty four hours after emergence, adult flies were sexed and placed into different cages until studied. The female : male ratio in the group mating was 1 : 5 and the males were marked by painting a dot on the thorax using different oil colours. Observation of mating behaviour was investigated every 30 minutes through 10-20 replications for all methods depending on the availability of flies. Data were analysed using ANOVA and the Student’s t-test, with significance demonstrated at the 95% confidence level. The results demonstrated that the frequency of contacts between males and females at different ages was a significantly different (p < 0.05) and that the duration of copulation was not significantly affected by fly age both method 1 (p > 0.05) and method 2 (p > 0.05). Copulation was only initiated following longer periods of contact, mainly in the range of 270-449 seconds. The highest frequency of copulation occurred between 7-8 days, but the duration of mating was similar between 5-8 days old. The study demonstrated that the methods developed were suitable for a mating compatibility study of C. bezziana. Key Words: Chrysomya bezziana, Mating Behaviour, Copulation, Myiasis
Geographical characteristics of Chrsyomya bezziana based on external morphology study Wardhana, April H.; Muharsini, S; Ready, P.D; Cameron, M.M; Hall, M.J.R
Indonesian Journal of Animal and Veterinary Sciences Vol 17, No 1 (2012)
Publisher : Indonesian Animal Sciences Society

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (119.56 KB) | DOI: 10.14334/jitv.v17i1.710

Abstract

Correct identification of Chrysomya bezziana is a fundamental step to evaluate the successfulness of the eradication program based on Sterile Insect Techinque (SIT). However, geographical variation of the fly is being controversial among scientists. The aim of the study was to investigate the effect of preservation method on visualisation of characters of external morphology and to analyse geographical variation of C. bezziana populations throughout their distribution regions. A total of 88 flies collected from 7 populations in Indonesia, 2 populations in Africa and each 1 population from Oman, India, Malaysia and Papua New Guinea (PNG) were tested in the study. All larvae were removed from natural myiasis cases. The larvae were reared at laboratory until they became adult flies. The samples were preserved into two methods, wet (80% ethanol) and dried (pin) methods. Ten external characters of head and body were observed.  Data were subjected to principal components and hierarchical cluster analyses in UNISTATÒ software. The Euclid distance measure was used for the cluster analysis, and the linking method used was the unweighted pair-group method using arithmetic average (UPGMA), to compute the distance between clusters. Results indicated that dried preservation (pinned samples) provided better external character than ethanol preservation. Based on external morphology of adult stages revealed that C. bezziana occurs as two geographical races, African and Asian races. There was no geographical variation of C. bezziana throughout Indonesian archipelago, except for the population from PNG forming its own cluster. Key Words: Myiasis, Chrysomya bezziana, Morphology, Geographical Variaton
Geographical characteristics of Chrsyomya bezziana based on external morphology study Wardhana, April H.; Muharsini, S; Ready, P.D; Cameron, M.M; Hall, M.J.R
Jurnal Ilmu Ternak dan Veteriner Vol 17, No 1 (2012): MARCH 2012
Publisher : Indonesian Animal Sciences Society

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (119.56 KB) | DOI: 10.14334/jitv.v17i1.710

Abstract

Correct identification of Chrysomya bezziana is a fundamental step to evaluate the successfulness of the eradication program based on Sterile Insect Techinque (SIT). However, geographical variation of the fly is being controversial among scientists. The aim of the study was to investigate the effect of preservation method on visualisation of characters of external morphology and to analyse geographical variation of C. bezziana populations throughout their distribution regions. A total of 88 flies collected from 7 populations in Indonesia, 2 populations in Africa and each 1 population from Oman, India, Malaysia and Papua New Guinea (PNG) were tested in the study. All larvae were removed from natural myiasis cases. The larvae were reared at laboratory until they became adult flies. The samples were preserved into two methods, wet (80% ethanol) and dried (pin) methods. Ten external characters of head and body were observed.  Data were subjected to principal components and hierarchical cluster analyses in UNISTATÒ software. The Euclid distance measure was used for the cluster analysis, and the linking method used was the unweighted pair-group method using arithmetic average (UPGMA), to compute the distance between clusters. Results indicated that dried preservation (pinned samples) provided better external character than ethanol preservation. Based on external morphology of adult stages revealed that C. bezziana occurs as two geographical races, African and Asian races. There was no geographical variation of C. bezziana throughout Indonesian archipelago, except for the population from PNG forming its own cluster. Key Words: Myiasis, Chrysomya bezziana, Morphology, Geographical Variaton
Evaluation of mating behaviour and mating compatibility methods for the Old World screwworm fly, Chrysomya bezziana. Wardhana, April H.; MM, Cameron; S, Muharsini; MJR, Hall
Jurnal Ilmu Ternak dan Veteriner Vol 18, No 4 (2013): DECEMBER 2013
Publisher : Indonesian Animal Sciences Society

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (237.362 KB) | DOI: 10.14334/jitv.v18i4.333

Abstract

The effectiveness of the Sterile Insect Technique program (SIT) to eradicate pest insects relies on the success of mating competitiveness between irradiated male flies and wild type males for the wild type females. It has been successfully applied for the New World screwworm fly (NWSF), Cochliomyia hominivorax but remains unproven for the Old World screwworm fly (OWSF), Chrysomya bezziana. The aim of the study was to develop methods for investigating mating behaviour and mating compatibility of C. bezziana under laboratory conditions. Two methods were used for studying mating: individual mating (method 1) and group mating (method 2). The flies used in this study were 5-7 days old. Twenty four hours after emergence, adult flies were sexed and placed into different cages until studied. The female : male ratio in the group mating was 1 : 5 and the males were marked by painting a dot on the thorax using different oil colours. Observation of mating behaviour was investigated every 30 minutes through 10-20 replications for all methods depending on the availability of flies. Data were analysed using ANOVA and the Student’s t-test, with significance demonstrated at the 95% confidence level. The results demonstrated that the frequency of contacts between males and females at different ages was a significantly different (p < 0.05) and that the duration of copulation was not significantly affected by fly age both method 1 (p > 0.05) and method 2 (p > 0.05). Copulation was only initiated following longer periods of contact, mainly in the range of 270-449 seconds. The highest frequency of copulation occurred between 7-8 days, but the duration of mating was similar between 5-8 days old. The study demonstrated that the methods developed were suitable for a mating compatibility study of C. bezziana. Key Words: Chrysomya bezziana, Mating Behaviour, Copulation, Myiasis
Molecular identification technique of Trypanosoma evansi by Multiplex Polymerase Chain Reaction Sawitri, Dyah H.; Wardhana, April H.; Wibowo, H.; Sadikin, M.; Ekawasti, Fitrine
Jurnal Ilmu Ternak dan Veteriner Vol 20, No 4 (2015): DECEMBER 2015
Publisher : Indonesian Animal Sciences Society

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (382.422 KB) | DOI: 10.14334/jitv.v20i4.1248

Abstract

Trypanosoma evansi is a Hemoflagella parasite that infects cattle and is known as the agents of Surra. Several other trypanosome species infects mammals: T. equiperdum, T. b. rhodesiense, T. b. gambiense, T. vivax, T. congolense, T.theileri. Some of these species is quite difficult to be distinguished morphologically with T. evansi through conventional techniques (thin blood smear). Molecular technique by polymerase chain reaction (PCR) is reported to have the ability to identify, characterize and diagnose trypanosomes accurately. However, a single PCR used is relatively expensive because it takes at least two or more pairs of primers to determine T. evansi. The purpose of this study is to develop T. evansi species identification techniques by multiplex PCR/mPCR (the three pairs of primer in one reaction) that takes the relatively fast and inexpensive. A total of 31 isolates T.evansi were obtained from Bblitvet Culture Collection (BCC) and the Department of Parasitology BBLitvet used in this study. Isolates represent isolates from endemic areas and Surra outbrake isolated from 1988-2014. DNA extraction performed on each sample, including Bang 87 isolates which has been purified as a positive control. Primers used are specific for T. evansi, the ITS-1, Ro Tat 1.2 VSG and ESAG 6/7. Before running mPCR, each primer is optimized by using a single PCR. The results showed that the three primers can be combined in a single reaction with mPCR technique and amplify each DNA fragment target perfectly, so identified 31 isolates as T. evansi. This technique can be applied in the field with a lower cost and faster time.Key Words: Trypanosoma evansi, Identification, Multiplex PCR
Evaluation of surra treatment strategies attacking horses and buffaloes in East Sumba District, Nusa Tenggara Timur Province of Indonesia (2010 – 2016) Dewi, Rita S.; Wardhana, April H.; Soejoedono, Retno D.; Mulatsih, Sri
Jurnal Ilmu Ternak dan Veteriner Vol 24, No 1 (2019): MARCH 2019
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (890.989 KB) | DOI: 10.14334/jitv.v24i1.1864

Abstract

Surra is a disease attacking livestock caused by a flagellated protozoan, Trypanosoma evansi. Indonesia archipelago is reported as an endemic country of the disease, except Sumba Island.  However, Surra outbreak occurred in this Island in 2010 due to livestock movement from the neighbour island, Sumbawa. It generated high mortality in livestock, particularly in horses and buffaloes. The aim of the study was to evaluate the effectiveness of Surra treatment strategies in East Sumba District from 2010-2016 and to estimate the incidence of Surra in the next few months (forecast). The treatment strategy of Surra in East Sumba was divided into 2 (two) periods e.g. the first period in 2010-2011 using Isomethamedium as the single drug (period I) and the second period in 2012 - 2016 using a combination between diminazene aceturate as curative and isomethamedium as a prophylactic drug (period II). All data in the present study was obtained from the local livestock agency of East Sumba District from 2010 – 2016 when Surra outbreak occurred. The effectiveness of those two treatment strategies was compared using the proportion test. The results demonstrated that morbidity and mortality of horses and buffaloes were significantly greater in the period I (2010-2011) compared to period II (2012-2016). The treatment strategy in the period II was able to decrease the proportion of morbidity in horses and buffaloes for 1.44% and 0.66%, respectively.  Likewise, the proportion of mortality in period II was also less than the period I from 3.79% to 1.30% for horses and from 2.80% to 0.55% for buffaloes. Based on forecasting study analysis using the control program projected with decomposition method for the next 12 months demonstrated that the treatment strategy in the period II could reduce the incidence and death of livestock by Surra. The treatment strategy using a combination between isometamedium and diminazene aceturate in East Sumba District might be more effective compared to using isometamedium alone. 
Genetic variability of ESAG6/7 gene Trypanosoma evansi Sawitri, Dyah Haryuningtyas; Wardhana, April H.
Jurnal Ilmu Ternak dan Veteriner Vol 22, No 1 (2017): MARCH 2017
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (387.524 KB) | DOI: 10.14334/jitv.v22i1.1638

Abstract

Trypanosoma evansi as an agent of Surra is one of the crucial parasitic diseases that cause great economic losses in Indonesia. These parasites need iron for growth and propagation phase which is obtained by receptor-mediated uptake of host transferin. The transferrin receptors are encoded by Expression Site Associated Genees (ESAGs). ESAG6/7 encodes transferrin receptors which reported have different affinities of a different host. The distinction of T. evansi pathogenicity is supposed to cause variability in the ESAG6/7 gene. This research was aimed to investigate the variability of genes ESAG6/7 T. evansi with different virulence in mice. This research was conducted in two steps: bioassay pathogeneicity in mice and analysis of ESAG6/7 gene sequences. The median survival time of mice was investigated after each group of mice infected by 25 T. evansi isolates from buffaloes where its geographically differ. The test results showed a difference of pathogenic virulence on 25 T. evansi isolates in mice. Sequence analysis of the ESAG6/7 gene from 25 T. evansi isolates origin from Indonesia tended to be homogeneous on the transferrin binding site but there was variability in the hypervariable site. These changes are able to separate high and low virulence of the T. evansi isolates. Phylogenetic tree analysis was formed 11 clades of 25 T. evansi. High virulence T. evansi was included in clades 7 and 10, while low virulence T. evansi was included in clade 5 and 11 and the moderate virulence was divided into those four clades.
Evaluation of surra treatment strategies attacking horses and buffaloes in East Sumba District, Nusa Tenggara Timur Province of Indonesia (2010 – 2016) Dewi, Rita S.; Wardhana, April H.; Soejoedono, Retno D.; Mulatsih, Sri
Jurnal Ilmu Ternak dan Veteriner Vol 24, No 1 (2019): MARCH 2019
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (890.989 KB) | DOI: 10.14334/jitv.v24i1.1864

Abstract

Surra is a disease attacking livestock caused by a flagellated protozoan, Trypanosoma evansi. Indonesia archipelago is reported as an endemic country of the disease, except Sumba Island.  However, Surra outbreak occurred in this Island in 2010 due to livestock movement from the neighbour island, Sumbawa. It generated high mortality in livestock, particularly in horses and buffaloes. The aim of the study was to evaluate the effectiveness of Surra treatment strategies in East Sumba District from 2010-2016 and to estimate the incidence of Surra in the next few months (forecast). The treatment strategy of Surra in East Sumba was divided into 2 (two) periods e.g. the first period in 2010-2011 using Isomethamedium as the single drug (period I) and the second period in 2012 - 2016 using a combination between diminazene aceturate as curative and isomethamedium as a prophylactic drug (period II). All data in the present study was obtained from the local livestock agency of East Sumba District from 2010 – 2016 when Surra outbreak occurred. The effectiveness of those two treatment strategies was compared using the proportion test. The results demonstrated that morbidity and mortality of horses and buffaloes were significantly greater in the period I (2010-2011) compared to period II (2012-2016). The treatment strategy in the period II was able to decrease the proportion of morbidity in horses and buffaloes for 1.44% and 0.66%, respectively.  Likewise, the proportion of mortality in period II was also less than the period I from 3.79% to 1.30% for horses and from 2.80% to 0.55% for buffaloes. Based on forecasting study analysis using the control program projected with decomposition method for the next 12 months demonstrated that the treatment strategy in the period II could reduce the incidence and death of livestock by Surra. The treatment strategy using a combination between isometamedium and diminazene aceturate in East Sumba District might be more effective compared to using isometamedium alone. 
Geographical characteristics of Chrsyomya bezziana based on external morphology study Wardhana, April H.; Muharsini, S; Ready, P.D; Cameron, M.M; Hall, M.J.R
Indonesian Journal of Animal and Veterinary Sciences Vol 17, No 1 (2012): MARCH 2012
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (119.56 KB) | DOI: 10.14334/jitv.v17i1.710

Abstract

Correct identification of Chrysomya bezziana is a fundamental step to evaluate the successfulness of the eradication program based on Sterile Insect Techinque (SIT). However, geographical variation of the fly is being controversial among scientists. The aim of the study was to investigate the effect of preservation method on visualisation of characters of external morphology and to analyse geographical variation of C. bezziana populations throughout their distribution regions. A total of 88 flies collected from 7 populations in Indonesia, 2 populations in Africa and each 1 population from Oman, India, Malaysia and Papua New Guinea (PNG) were tested in the study. All larvae were removed from natural myiasis cases. The larvae were reared at laboratory until they became adult flies. The samples were preserved into two methods, wet (80% ethanol) and dried (pin) methods. Ten external characters of head and body were observed.  Data were subjected to principal components and hierarchical cluster analyses in UNISTATÒ software. The Euclid distance measure was used for the cluster analysis, and the linking method used was the unweighted pair-group method using arithmetic average (UPGMA), to compute the distance between clusters. Results indicated that dried preservation (pinned samples) provided better external character than ethanol preservation. Based on external morphology of adult stages revealed that C. bezziana occurs as two geographical races, African and Asian races. There was no geographical variation of C. bezziana throughout Indonesian archipelago, except for the population from PNG forming its own cluster. Key Words: Myiasis, Chrysomya bezziana, Morphology, Geographical Variaton