Detection of Foot-and-Mouth Disease Virus in the Absence of Clinical Disease in Cattle and Buffalo in South East Asia.

Foot-and-mouth disease virus (FMDV) is widespread throughout much of the world, including parts of South East Asia. Surveillance is often limited in endemic areas, relying predominantly on passive outbreak reporting. As part of the World Organisation for Animal Health (OIE)'s South East Asia and China Foot-and-Mouth Disease Project (SEACFMD), field sampling was performed to help understand evidence of widespread virus exposure observed in previous studies. Serum and dry mucosal swabs were collected to evaluate the presence of FMDV RNA on the nasal, oral, and dorsal nasopharyngeal mucosal surfaces of 262 healthy cattle (n = 84 in Laos; n = 125 in Myanmar) and buffalo (n = 48 in Laos; n = 5 in Myanmar) immediately following slaughter in three slaughterhouses. Swabs and serum were tested by the OIE/FAO World Reference Laboratory for foot-and-mouth disease (WRLFMD) using pan-serotypic real-time reverse transcription-PCR (rRT-PCR) and serum was evaluated using the FMD PrioCHECK non-structural protein (NSP) ELISA. In total, 7.3% of animals had detectable FMDV RNA in one or more of the three sites including 5.3% of nasopharyngeal swabs, 2.3% of oral swabs, and 1.5% of nasal swabs. No FMDV RNA was detected in serum. Overall, 37.8% of animals were positive for NSP antibodies, indicating likely past natural exposure to FMDV. Results were comparable for Laos and Myanmar, and for both cattle and buffalo, and were not significantly different between age groups. Detectable FMDV RNA present on the oral and nasal mucosa of clinically-healthy large ruminants in Laos and Myanmar demonstrates the importance of sampling asymptomatic animals as part of surveillance, and may indicate that subclinical infection plays a role in the epidemiology of FMD in these countries.

Evaluating the utility of national-scale data to estimate the local risk of foot-and-mouth disease in endemic regions.

Knowledge of the distribution of foot-and-mouth disease (FMD) is required if control programmes are to be successful. However, data on the seroprevalence and incidence of affected villages in developing countries with endemic disease are scarce. This is partly due to resource constraints as well as the logistical challenges of conducting intensive surveys and diagnostic testing in remote locations. In this study, we evaluated the performance of low resolution national-scale data against high resolution local survey data to predict the FMD serological status of 168 villages in the Mandalay and Sagaing Regions of central Myanmar using both logistic regression and random forest modelling approaches. Blood samples for ELISA testing were collected from approximately 30 cattle per village in both the 6 to 18 month age range and in the over 18 month age range to distinguish between recent and historical exposure, respectively. The results of the animal level tests were aggregated to the village level to provide the outcome of interest (village positive or not positive for FMD), and three explanatory data sets were constructed: using only nationally available data, using only data collected by survey and using the combined survey and nationally available data. The true seroprevalence of FMD at the village level was 61% when only young animals were included, but increased to 87% when all animals were included. The best performing model was a logistic regression model using the combined national and survey data to predict recent infection in villages. However, this still incorrectly classified 40% of villages, which suggests that using national-level data were not reliable enough for extrapolating seroprevalence in regions where conducting detailed surveys is impractical. Other methods for collected data on FMD such as the use of local reporting should be explored.

Estimating foot-and-mouth disease (FMD) prevalence in central Myanmar: Comparison of village headman and farmer disease reports with serological findings.

The impacts of foot-and-mouth disease (FMD) on food security in developing countries are difficult to quantify due to the scarcity of accurate data on the prevalence and incidence of affected villages. This is partly due to resource constraints as well as the logistical challenges of conducting regular diagnostic testing in remote locations. In this study, we used descriptive analysis and latent class analysis (LCA) models to analyse data collected during a field survey of 160 villages in central Myanmar in the Mandalay and Sagaing Regions over the 2012-2016 time period. We evaluated the performance of verbal reports made by village householders and headmen against serological data to retrospectively determine the FMD-infection status of our study area and to identify factors contributing to under-reporting. Blood samples were collected from approximately 30 cattle per village in both the 6- to 18-month age range and over 18-month age range to distinguish between recent and historic exposure. Village householders were asked to identify pictures of FMD-affected cattle amongst pictures of cattle affected with other common endemic diseases to assess the accuracy of their verbal reporting. The serological results confirmed that FMD is endemic in central Myanmar with village-level seroprevalence estimated at 56% for animals 6-18 months of age and 80% when all age groups were considered together. Most village householders were familiar with the clinical signs of FMD-affected cattle (72%). Based on the results from the LCA models, the village headman had a sensitivity of 77% and specificity of 75% for identifying FMD outbreaks in their village, whereas individual householders had a higher sensitivity and lower specificity of 80% and 56%, respectively. The level of disagreement between the different sources was correlated with the total number of cattle in the village and may potentially be worse in villages where endemic FMD may have led to a high level of natural immunity in cattle and subsequent masking of clinical signs. However, other regional effects such as the intensity of FMD extension efforts cannot be ruled out. Overall, the results suggest that verbal reports of FMD outbreaks from village headmen may be a useful tool to integrate into active FMD surveillance programmes in developing countries.

Genome Sequences of Foot-and-Mouth Disease Viruses of Serotype O Lineages Mya-98 and Ind-2001d Isolated from Cattle and Buffalo in Myanmar.

We report whole-genome sequences and partial genome sequences of eight foot-and-mouth disease viruses obtained from outbreak samples in Myanmar in 2016 and 2017. These viruses are classified into O/ME-SA/Ind-2001d and O/SEA/Mya-98 lineages.

Comparative analysis of the methanogen diversity in horse and pony by using mcrA gene and archaeal 16s rRNA gene clone libraries.

Comparative analysis of methanogen compositions in the feces of horse and pony was carried out by constructing the α -subunit of methyl coenzyme-M reductase (mcrA) gene and 16S ribosomal RNA gene (16S rRNA) clone libraries. The mcrA clone library analysis indicated that Methanomicrobiales was predominant in both horse and pony. Furthermore, most of the clones of the 16S rRNA gene library showed that Methanomicrobiales was also predominant in horse and pony, but the LIBSHUFF analysis showed that the horse and pony libraries were significantly different (P < 0.05). Most of operational taxonomic units (OTUs) showed low similarity to the identified methanogens in both the mcrA and the 16S rRNA clone libraries. The results suggest that horse and pony harbor unidentified and novel methanogens in their hindgut. The methanogen population was higher in horse than in pony; however, the anaerobic fungal population was similar in horse and pony. The methanogen diversity was different between two breeds of Equus caballus.

Ruminal fermentation and microbial ecology of buffaloes and cattle fed the same diet.

Although buffaloes and cattle are ruminants, their digestive capabilities and rumen microbial compositions are considered to be different. The purpose of this study was to compare the rumen microbial ecology of crossbred water buffaloes and cattle that were fed the same diet. Cattle exhibited a higher fermentation rate than buffaloes. Methane production and methanogen density were lower in buffaloes. Phylogenetic analysis of Fibrobacter succinogenes-specific 16S ribosomal RNA gene clone library showed that the diversity of groups within a species was significantly different (P < 0.05) between buffalo and cattle and most of the clones were affiliated with group 2 of the species. Population densities of F.succinogenes, Ruminococcus albus and R. flavefaciens were higher until 6 h post-feeding in cattle; however, buffaloes exhibited different traits. The population of anaerobic fungi decreased at 3 h in cattle compared to buffaloes and was similar at 0 h and 6 h. The diversity profiles of bacteria and fungi were similar in the two species. The present study showed that the profiles of the fermentation process, microbial population and diversity were similar in crossbred water buffaloes and crossbred cattle.

Comparative analysis of methanogen diversity in the rumen of crossbred buffalo and cattle in the Philippines by using the functional gene mcrA.

Comparative analyses of methanogen diversity in the rumen of crossbred buffalo and cattle fed the same diet in the Philippines was performed by cloning the methyl coenzyme M reductase A (mcrA) gene. The cattle and buffalo libraries consisted of 50 clones each. Comparative analysis of the amino acid sequence revealed that these 2 libraries differed significantly (P < 0.01). The deduced amino acid sequences of the clones were classified into 9 operational taxonomic units (OTUs) in buffalo and 11 OTUs in cattle. Sequence similarity between the clones and known cultured methanogens ranged from 86 to 97 % for buffalo and 84 to 99 % for cattle. Methanobrevibacter species were predominant in buffalo (64 % of the clones), and an unknown mcrA was predominant in cattle (52 % of the clones). A large number of clones with low similarity to cultivated methanogens was observed in both buffalo and cattle, suggesting the presence of an unknown methanogen species in their rumen.

Real-time PCR assays for monitoring anaerobic fungal biomass and population size in the rumen.

The relationship between copy numbers of internal transcribed spacer 1 (ITS1) and biomass or zoospore count of anaerobic fungi was studied to develop a quantitative real-time PCR-based monitoring method for fungal biomass or population in the rumen. Nine fungal strains were used to determine the relationship between ITS1 copy number and fungal biomass. Rumen fluid from three sheep and a cow were used to determine the relationship between ITS1 copy number and fungal population. ITS1 copy number was determined by real-time PCR with a specific primer set for anaerobic fungi. Freeze-dried fungal cells were weighed for fungal biomass. Zoospore counts were determined by the roll-tube method. A positive correlation was observed between both ITS1 copy number and dry weight and ITS1 copy number and zoospore counts, suggesting that the use of ITS1 copy numbers is effective for estimating fungal biomass and population density. On the basis of ITS1 copy numbers, fluctuations in the fungal population in sheep rumen showed that although the values varied among individual animals, the fungal population tended to decrease after feeding. In the present study, a culture-independent method was established that will provide a powerful tool for understanding the ecology of anaerobic fungi in the rumen.