Novel MHC BLB2 gene polymorphism and its association with IgY concentration and Newcastle disease antibody titer in IPB-D2 chickens.

This study aimed to identify the polymorphism of the B Locus Beta 2 (BLB2) gene and its association with immunoglobulin Y (IgY) concentration and Newcastle disease (ND) antibody titer; we analyzed BLB2 gene expression in different categories of ND antibody titers in IPB-D2 chickens. The total sample used was 100 IPB-D2 chickens. Blood samples were collected at 21 weeks old for an ELISA (enzyme-linked immunoassay) test, an HI (hemagglutination inhibition) test, and genotyping. The method for BLB2 polymorphism was Sanger sequencing. Analysis of BLB2 gene expression was performed using the cecal tonsil tissue of IPB-D2 chickens. Polymorphism data were analyzed using SNPstats and DNAsp (DNA Sequence Polymorphism) software. The association of the single-nucleotide polymorphisms (SNPs) with IgY concentration and ND antibody titer was analyzed using SAS software (version 9.2). The genotype mean values were compared by means of a T test. The relative mRNA expression analysis was performed using a quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that 13 SNPs were found in exon 2 and exon 3 in the BLB2 gene. As many as 4 out of the 13 SNPs were associated with IgY concentration. As many as 9 out the 13 SNPs may have changed amino acids. The ΔCt value showed that the expression of the BLB2 gene in IPB-D2 chickens with high ND antibody titers is higher than IPB-D2 chickens with low ND antibody titers. In conclusion, the AA genotype of g.458 T > A was associated with high IgY concentrations, and the BLB2 gene presented with a high expression in IPB-D2 chickens with high ND antibody titers.

The diversity and quality of forages and their potency as herbal anthelmintic for swamp buffalo in Brebes District, Central Java.

Background and Aim: Swamp buffaloes play an important role in the rural economy of Indonesia. They consume various forages during their grazing time, including those with anti-parasitic potential. However, the information about the type and quality of forages and their potential as a natural anthelmintic for swamp buffalo is very limited. This study aimed to identify the diversity, quality, and anthelmintic potency of forages consumed by swamp buffaloes reared in Bantarkawung Subdistrict, Brebes District, Central Java Province, Indonesia. Materials and Methods: Samples of forages were obtained during three observation periods of the study, with a 12-week interval between each period. Forage diversity was evaluated by identifying its consumption by swamp buffaloes during their grazing activity in the field and feeding time in their shed. The quality of forages was analyzed using proximate analysis to measure their dry matter (DM), crude fiber (CF), crude protein (CP), crude fat (CFat), and ash contents. This is followed by the calculation of their total digestible nutrient based on the proximate analysis results. Botanical composition analysis was then conducted to measure the predominance of forages consumed by the livestock during their grazing activity. Literature reviews were carried out to explore forage's anthelmintic activity. Results: The results showed that swamp buffaloes consume nine species of forage in the shed and 47 in the grazing area, including nine legumes, 18 grass, and 20 others. Swamp buffaloes consumed forages of lower quality, which contained high CF contents and varying levels of other nutrients below their daily nutritional needs. The grazing activity allowed swamp buffaloes to consume a higher variety of forages with better nutritional quality, thereby enabling them to meet their nutritional needs. Legumes and other forages served as the major protein sources, providing CP of 20.03% DM and 11.53% DM, and CF levels of 17.01% DM and 20.35% DM, respectively. The results also showed that the consumption of these forages increased during the rainy season. The predominant species of legumes consumed were Leucaena leucocephala and Acacia spp., while Alternanthera sessilis and Merremia umbellata were the predominant species of other forages. A total of 13 of the 47 species could potentially be used as natural anthelmintic due to their secondary metabolites, namely, tannin, flavonoid, saponin, terpenoid, diterpenoid, and mimosine. These compounds exert anthelmintic effects by inhibiting egg-hatching and larval development, as well as damaging the surface structure of both larvae and adult worms, ultimately leading to the death of the parasites. Conclusion: Overall, swamp buffaloes consumed more variety of forages during grazing compared to when they were kept in sheds. While the low-variety and low-quality forages provisioned for swamp buffaloes in their shed resulted in a low nutrient intake below their daily requirement. Furthermore, daily grazing activities allowed swamp buffaloes to fulfill and supplement their need by consuming a variety of grasses, legumes, and other forages in their respective grazing areas. Some of these forages also have the potential to become natural anthelmintic because they contain secondary metabolites, such as tannins, flavonoids, saponins, terpenoids, diterpenoids, and mimosine.

Flow cytometric evaluation of CD4+ and CD8+ T-cell in IPB-D2 chickens with different Newcastle disease antibody titers level.

Background and Aim: IPB-D2 chickens are selected from IPB-D1 due to their disease-resistance characteristics. One-way to evaluate the strength of a chicken's immune system is by examining the number of circulating T lymphocytes. This assessment can be conducted using a modern analytic method called flow cytometry which relies on monoclonal antibodies to detect the relative proportions of each cell and measure the quality and quantity of biological and physical features of cells, including specific membrane or intracellular glycoprotein markers. Therefore, this study aimed to evaluate the population of lymphocytes, cluster of differentiation (CD)4+ and CD8+ in IPB-D2 chickens. Materials and Methods: Flow cytometry was used to evaluate the population of lymphocytes, CD4+, and CD8+ in IPB-D2 chickens. The data obtained in this study were analyzed by Minitab, and the mean values were compared using a t-test. Results: The lymphocytes, CD4+, and CD8+ populations of IPB-D2 chicken with high Newcastle disease (ND) antibody titers were 65.04%, 10.53%, and 5.47%. Meanwhile, this breed, with low ND antibody titers had lymphocytes CD4+ and CD8+ population of 57.19%, 8.40%, and 4.11 %. The comparison of CD4+ and CD8+ populations in chickens with high and low ND antibody titers was 1.92 and 2.04, respectively. Conclusion: IPB-D2 chickens with high ND antibody titers exhibited increased lymphocyte, CD4+, and CD8+ cell populations in comparison to those with low ND antibody titers. However, the high ND antibody titer group had a lower CD4+/CD8+ ratio.

Seasonal dynamics of Fasciola gigantica transmission in Prafi district, Manokwari Regency, West Papua, Indonesia.

Background and Aim: Indonesia's farming practices are a perfect setting for establishing an infection with Fasciola gigantica which can result in economically detrimental. The objectives of the current study were to describe and analyze the transmission dynamics of fasciolosis (F. gigantica) in Prafi District, to provide information on effective control strategies and to identify risk factors associated with fasciolosis in cattle. Materials and Methods: Fecal samples were purposively collected from the rectum of 60 beef cattle in Prafi District, Manokwari Regency, West Papua Province, Indonesia. The samples were collected once a month for 8 months from April 2019 to November 2019. Furthermore, the samples were taken from two rearing system groups: 30 stall cattle and 30 cattle in a palm oil garden. The presence of F. gigantica eggs in the fecal samples was examined using a modified Danish Bilharziasis Laboratory technique-sedimentation. Meanwhile, the antigenic diagnosis of Fasciola in the fecal samples was analyzed using an enzyme-linked immunosorbent assay kit to perform an indirect sandwich assay on feces. Snails were collected from an irrigation canal, rice field, and palm oil garden canal around Prafi District. These snails were examined for infection with cercariae larvae of F. gigantica by cercarial shedding and crushing techniques. Results: The peak occurrence of F. gigantica infection was identified in August (65.00%) and the lowest in June (35.00%). The highest prevalence of fasciolosis in cattle was recorded in August and November (90.00%) and the lowest was in May (40.00%). Moreover, the highest prevalence of fasciolosis in cattle exposed to the palm oil garden was recorded in April (53.33%) and the lowest prevalence of F. gigantica infection was recorded in June (23.33%). In total, 2046 snails were screened by the cercarial shedding and crushing method; of these, 426 (20.82%) were found to be positive for trematode cercariae. The prevalence of F. gigantica infection in Lymnaea spp. snails was highest in November (47.46%) and lowest in April (9.28%). Conclusion: The current study shows that beef cattle grown in two types of rearing systems in Prafi District were infected with F. gigantica during the dry and rainy season. It was revealed that Lymnaea spp. are common snails found in and around Prafi District, and can act as intermediate hosts with an infective stage of trematode. The transmission to cattle was highly effective, despite the short activity period, the low infection rate of snails, and the incidental anthelminthic treatment.

Isolation and molecular characterization of fowl aviadenovirus associated with inclusion body hepatitis from poultry in Banten and West Java, Indonesia.

BACKGROUND AND AIM: Fowl avidenoviruses (FAdVs) are generally considered ubiquitous, but certain serotypes and strains are known to be associated with primary diseases, such as inclusion body hepatitis (IBH). Since 2018, the outbreak of IBH has been reported in part provinces of Indonesia. This study aimed to isolate and molecularly characterize the FAdV from Banten and West Java Provinces of Indonesia and described the phylogenetic relationship with the FAdV that has been characterized in other countries. MATERIALS AND METHODS: A total of 25 FAdV archive samples have been collected from January to August 2019 from clinical cases of FAdV infection in Banten and West Java Provinces, Indonesia. Collected samples were inoculated in 10-day-old specific-pathogenic-free chicken embryonated eggs. Hexon gene of FAdV was detected using polymerase chain reaction (PCR) with a primer set from previous study. To gain a better understanding of the FAdV genetic properties and construct the phylogeny tree, the PCR products were sequenced and subjected to a BLAST search and inferred using the neighbor-joining method by bootstrap test 1000×. RESULTS: FAdV-D and FAdV-E are present in Banten, Indonesia. The phylogenetic analysis of 850 nucleotides that encode 289 amino acid of the partial hexon gene shows that the isolates Broiler/MSL/Ciputat-149/18, Broiler/MSL/Lebak-151/18, and Broiler/MSL/Ciputat-29/19 have 100% homology with FAdV-E TR/BVKE/R/D-1 from Turkey, whereas the isolates Layer/MSL/Ciputat-20/19 and Broiler/MSL/Ciputat-30/19 have 100% homology with FAdV-D strain 685 from Canada. CONCLUSION: The present study provides updates of the circulating FAdV in commercial poultry flocks in Banten and West Java Provinces, Indonesia. Since the FAdV vaccine was unavailable in Indonesia, this result might be used as guidance to select a proper FAdV vaccine strain. Our result indicates that at least two FAdV species were circulating among poultry in Banten and West Java Provinces, Indonesia; they are FAdV-D and FAdV-E.

The contribution of domestic animals to the transmission of schistosomiasis japonica in the Lindu Subdistrict of the Central Sulawesi Province, Indonesia.

BACKGROUND AND AIM: Schistosomiasis is endemic in Indonesia and is found in three remote areas in Central Sulawesi Province. Non-human mammals serve as reservoir hosts, meaning the disease is zoonotic. The previous schistosomiasis studies in animals from the Lindu Subdistrict did not determine which domestic animal species can serve as the primary source of transmission. No animals have been treated in Indonesia to control the disease; therefore, the parasite's life cycle is not blocked entirely. This study aimed to determine the prevalence and identify the risk factors associated with, Schistosoma japonicum infection in animals, and identify animals' relative contributions to S. japonicum transmission in the Lindu Subdistrict. MATERIALS AND METHODS: A cross-sectional survey of S. japonicum infected animals was conducted in five villages of the Lindu Subdistrict. Fecal samples were collected from 134 selected animals (13 cattle, 26 buffaloes, 28 horses, 59 pigs, and 8 dogs). S. japonicum infection and infection intensity were determined using the Danish Bilharziasis Laboratory method. Environmental contamination with schistosome eggs was measured. The data were analyzed using a Chi-square test. RESULTS: The overall prevalence of schistosomiasis was 32.9%, with the prevalence of infection in each species of animal at 61.5% in cattle, 42.3% in buffaloes, 25.0% in horses, 35.6% in pigs, and 12.5% in dogs. Free-range pigs were 8.667 times more likely to have S. japonicum infection than pigs kept in cages. Buffaloes, cattle, and horses were the primary sources of S. japonicum egg contamination, with relative transmission indices of 59.15%, 22.80%, and 10.61%, respectively. CONCLUSION: Bovines and horses are the main contributors to schistosomiasis transmission in the Lindu Subdistrict. In conjunction with other schistosomiasis control programs, the government should treat infected animals living within endemic areas where there are high infection rates of S. japonicum.

Prevalence and risk factors of trematode infection in swamp buffaloes reared under different agro-climatic conditions in Java Island of Indonesia.

BACKGROUND AND AIMS: This study was conducted to estimate the prevalence and intensity and to identify the associated risk factors and impact of trematode infection in swamp buffaloes reared under different agro-climatic conditions in Java, Indonesia. MATERIALS AND METHODS: A total of 580 fecal samples were collected from swamp buffaloes in five different agro-climatic areas in Banten and Central Java Provinces, Indonesia. The fecal samples were examined using the Danish Bilharziasis Laboratory Technique to determine the prevalence and intensity of trematode infection. The risk factors for infection were determined from an in-depth interview of owners/keepers, and the results were analyzed using Chi-square tests and multiple logistic regression. The infection was also correlated with swamp buffalo production parameters (body weight and body condition score [BCS]). RESULTS: From all fecal samples, the overall prevalence of trematode infection was 64.83%, which comprised Fasciola spp. (16.03%; mean eggs per gram [EPG]±SD: 1.02±0.43) and Paramphistomatidae (62.93%; mean EPG±SD: 1.01±0.66). The main risk factor for trematode infection was feeding animals with rice straw (odds ratio [OR]: 40.124); the risk of trematode infection was 40.142 times higher in buffaloes that consumed rice straw. Other risk factors included the frequency of anthelmintic treatment (OR: 4.666), age (OR: 0.449), and drinking water source (OR: 0.358). Trematode infection did not significantly affect the body weight or BCS of swamp buffaloes. CONCLUSION: Although the prevalence of trematode infection was high in swamp buffaloes, the intensity of infection was low, and the infection did not affect the animals' physical parameters.

Efficacy of a recombinant HVT-H5 vaccine against challenge with two genetically divergent Indonesian HPAI H5N1 strains.

The swift evolution rate of avian influenza (AI) H5N1 virus demands constant efforts to update inactivated vaccines to match antigenically with the emerging new field virus strains. Recently, a recombinant turkey herpesvirus (rHVT)-AI vaccine, rHVT-H5, expressing the HA gene of a highly pathogenic avian influenza (HPAI) H5N1 clade 2.2 A/Swan/Hungary/499/ 2006 strain inserted into FC-126 strain of HVT vector, has been developed to combat current threats in poultry industry. Here, we present the results of two trials where rHVT-H5 was tested alone or in combination with inactivated H5N1 vaccines (the latter vaccines contained antigens produced by using a clade 2.1.3 HPAI H5N1 virus [A/Ck/WestJava-Nagrak/2007] in the first trial or mixture of antigen produced by strain A/Ck/WestJava-Nagrak/2007 and A/Ck/Banten-Tangerang/2010 [bivalent vaccine] for second trial) in broiler chickens (Gallus gallus domesticus) carrying maternally derived antibodies to H5N1 and then challenged with Indonesian HPAI H5N1 field isolates. The effectiveness of vaccination was evaluated on the basis of clinical protection (morbidity and mortality) and measurement of virus shedding after challenge. Immune response to vaccination was followed by serology. In the first experiment, chickens were vaccinated at the day of hatch with rHVT-H5 alone (Group 1) or combined with inactivated vaccine at day old (Group 2) or at 10 days of age (Group 3). The chickens along with nonvaccinated hatch-mates were challenged at 28 days of age with the HPAI H5N1 field isolate dade 2.1.3 A/Chicken/WestJava-Subang/29/2007. Eighty, 100%, and 80% clinical protection was recorded in Group 1, 2, and 3, respectively. A similar experiment was performed a second time, but the chicks in Group 3 received the inactivated vaccine earlier, at 7 days of age. Challenge was performed at 28 days of age using a different H5N1 isolate, clade 2.1.3 A/Ck/Purwakarta-Cilingga/142/10. Clinical protection achieved in the second trial was 95%, 75%, and 90% in Group 1, 2, and 3, respectively. Shedding of challenge virus was significantly lower in the vaccinated groups compared with controls in both experiments. Vaccinated birds developed hemagglutination inhibition antibody response to H5N1 by the time of challenge. These experiments confirmed that the rHVT-H5 vaccine applied alone or in combination with inactivated H5N1 vaccines could provide high level (> 80%) clinical protection against divergent HPAI H5N1 field isolates after single immunization by 4 wk of age and a significant reduction in the excretion of challenge virus.

Complementary monoclonal antibody-based dot ELISA for universal detection of H5 avian influenza virus.

BACKGROUND: Rapid diagnosis and surveillance for H5 subtype viruses are critical for the control of H5N1 infection. RESULTS: In this study, H5 Dot ELISA, a rapid test for the detection of avian H5N1 influenza virus, was developed with two complementary H5 monoclonal antibodies. HA sequencing of escape mutants followed by epitope mapping revealed that the two Mabs target the epitope component (189th amino acid) on the HA protein but are specific for different amino acids (189Lys or 189Arg). Gene alignment indicated that these two amino acids are the most frequent types on this position among all of the H5 AIV reported in GeneBank. These two H5 Mabs were used together in a dot ELISA to detect H5 viral antigen. The detection limit of the developed test for multiple clades of H5N1 viruses, including clades 0, 1, 2.1, 2.2, 2.3, 4, 7, and 8, was less than 0.5 hemagglutinin units. The specificity of the optimized dot ELISA was examined by using 100 H5 strains, including H5N1 HPAI strains from multiple clades, 36 non-H5N1 viruses, and 4 influenza B viruses. No cross-reactivity was observed for any of the non-H5N1 viruses tested. Among 200 random poultry samples, the test gave 100% positive results for all of the twelve RT-PCR-positive samples. CONCLUSIONS: Considering that the test is convenient for field use, this H5 Dot ELISA can be used for on-site detection of H5N1 infection in clinical or environmental specimens and facilitate the investigation of H5N1 influenza outbreaks and surveillance in poultry.

An inactivated H5N2 vaccine reduces transmission of highly pathogenic H5N1 avian influenza virus among native chickens.

Vaccination against H5N1 highly pathogenic avian influenza in endemically affected areas is a potentially attractive option for local prevention and control. In Indonesia the majority of local outbreaks have occurred in back yard flocks with native chickens, and it is therefore of interest to determine whether these birds can be protected against infection by vaccination. To this end two transmission experiments were carried out with H5N1 virus (A/chicken/Legok/2003) in vaccinated and unvaccinated native chickens. The vaccine contained an inactivated heterologous H5N2 strain (A/turkey/England/N28/73 H5N2). Birds were vaccinated at 4 and 7 weeks of age and challenged at 10 weeks of age. During 10 days post-challenge tracheal and cloacal swabs were taken for virus isolation, and serum blood was collected regularly to measure haemaglutinin inhibiting (HI) antibody responses. The results show that transmission of H5N1 virus was rapid and efficient in unvaccinated birds, that infection and transmission were completely prevented in vaccinated birds, and that vaccinated birds that were exposed to unvaccinated inoculated birds were still protected from infection. These findings indicate that vaccination with a heterologous H5N2 vaccine is able to prevent virus transmission in flocks of native chickens.