Are infectious reproductive pathogens of large ruminants a threat to improving food security? An investigation from Cambodia.

In Cambodia, information on common pathogens causing reproductive losses in cattle and buffalo are lacking, despite there being a need to address livestock health to enhance food security. We analysed stored buffalo (n = 29) and cattle (n = 471) serum samples collected in 2016 using commercially available enzyme-linked immunosorbent assay kits. Antibodies to Neospora caninum, bovine viral diarrhoea virus (BVDV), Leptospira interrogans serovar Hardjo and Brucella abortus were detected in buffalo samples at 79.3% (95% CI 64.6-94.0), 3.4% (95% CI 0-10.0), 0% and 0%, respectively, and in cattle at 4.2% (95% CI 2.4-6.0), 6.4% (95% CI 4.2-8.6), 8.1% (95% CI 5.6-10.6) and 0%, respectively. The high N. caninum seroprevalence in buffalo was associated with increasing age, with buffalo having a 13.1% chance of being seropositive at birth, increasing to 99.4% by age 7 (p = 0.045). This suggests a predominance of horizontal transmission, possibly from exposure to faeces from free-roaming village dogs. Cattle L. interrogans serovar Hardjo seroprevalence was highest in Tbong Khmum province (20.4%) compared to other provinces (p < 0.001), and may be compromising bovine fertility and creating a zoonotic risk for smallholders who may contract leptospirosis from farm work. These high infection rates prompt further research to determine: to what extent these pathogens are linked to the low reproductive efficiency observed in large ruminants in Cambodia, the risk factors to pathogen exposure and appropriate strategies to reduce these risks. Low BVDV and B. abortus exposure is an important observation. Increasing large ruminant livestock trade into the country will require improved biosecurity and disease surveillance to prevent their emergence. An enhanced understanding of the status of infectious reproductive livestock pathogens in Cambodia can assist development projects to make evidence-based strategies to enhance cattle and buffalo health and improve food security.

Which species is in the faeces at a time of global livestock movements: single nucleotide polymorphism genotyping assays for the differentiation of Fasciola spp.

Fasciolosis, caused by Fasciola hepatica and Fasciola gigantica, is a globally distributed zoonotic disease of livestock. While F. hepatica and F. gigantica have temperate and tropical distributions, respectively, parasite sympatry occurs in parts of Asia and Africa. A growing protein demand has the potential to facilitate the translocation of parasites from endemic to non-endemic areas, via associated international livestock movements. Such is the case in Southeast Asia, where livestock trade from F. hepatica-endemic countries into China and Vietnam may account for detection of F. hepatica hybrid/introgressed forms. Of particular importance is Lao People's Democratic Republic, which acts as a major livestock thoroughfare for the region. Our ability to understand the impacts of livestock-associated Fasciola spp. movements on local animal and human health is hindered by a lack of ante-mortem diagnostic tools allowing species differentiation. Molecular tools have been developed for Fasciola spp. differentiation, however those rely on access to pure DNA from adult specimens, limiting their application to post-mortem use. Our aim was to detect and differentiate F. hepatica from the endemic F. gigantica in local smallholder cattle in a region of Southeast Asia with frequent livestock trafficking. To do this we designed and validated ante-mortem molecular assays for Fasciola spp. differentiation targeting single-nucleotide polymorphisms (SNPs) within ITS1 and lsrRNA. We then deployed these SNP genotyping assays to diagnose Fasciola spp. infection in 153 local cattle from 27 villages in Northern Laos. We demonstrate the presence of F. hepatica DNA, confirmed by qualitative Sanger and quantitative Illumina amplicon sequencing of ITS1 and lsrRNA, and highlight the shortfalls of Sanger sequencing for Fasciola spp. identification due to the preferential amplification of F. gigantica nucleotides in mixed DNA samples. The outlined protocol enables rapid surveillance of faecal samples for the presence of Fasciola species eggs, their co-infection and/or infection with F. hepatica/F. gigantica hybrids.

A quick and simple benchtop vortex egg-disruption approach for the molecular diagnosis of Fasciola hepatica from ruminant faecal samples.

Commonly employed diagnostic methods for Fasciola spp., such as a traditional sedimentation and faecal egg count, or a commercially available coprological ELISA, have limitations in their sensitivity or ability to differentiate species. A reliable DNA isolation method coupled with real-time PCR addresses these issues by providing highly sensitive and quantitative molecular diagnosis from faecal samples. The current study evaluated a standard benchtop vortex for F. hepatica egg disruption in sheep and cattle faecal samples and determined the minimum faecal egg load required for a positive result from un-concentrated (raw) faecal samples. The minimum faecal egg load for a positive real-time PCR result from 150 mg raw faecal sample was 10 and 20 eggs per gram for sheep and cattle, respectively. No significant difference (P = 0.4467) between disruptions on a benchtop vortex for 5 or 10 min was observed when compared to 40 s of disruption at 6.0 m/s in a benchtop homogeniser.

Livestock and livelihoods of smallholder cattle-owning households in Cambodia: the contribution of on-farm and off-farm activities to income and food security.

The majority of smallholder farming households in Cambodia are rurally based and rely on agriculture to support their livelihoods. However, in recent years, growth in the agriculture sector has stagnated with farmers facing several challenges including declining prices for traditional crops and irregular rainfall patterns. This has led to a need for farmers to diversify income sources with livestock promoted as a more viable livelihood activity, particularly the raising of cattle and poultry. However, uncertain profitability of livestock activities is a common perception by smallholders, especially where animals have not been traditionally viewed as a primary income source. To address this, information is required which compares the income and expenses associated with livestock raising to other on-farm activities and off-farm sources. This study reports on a survey of livelihood survey of 17 male and 21 female representatives of 20 households owning cattle in Cambodia, comparing the associated income and expenses of the various livelihood activities in the 12-month period from January to December 2016. Combined total household income from both on-farm and off-farm sources ranged from USD875 to 17730 with an average of USD6779, representing 51% and 49% from on-farm and off-farm activities, respectively. Cattle raising was the most common source of on-farm income (85%), contributing USD1064 and representing 22% of total household income. General household expenses, such as food and transportation (including expenses associated with off-farm employment), represented the majority of total household expenses (79%). Gross profit calculations indicated higher values for pig raising (USD1841.79), cattle (USD950.80) and non-rice crops (USD884) whilst the highest gross margin value was recorded for cattle (89.33%) followed by vegetables (85.27%) and non-rice crops (83.08%). Whilst wet season and dry season rice returned a negative gross profit value of USD197.27 and USD90.60 on average per household, respectively, both were ranked as most important for household consumption, followed by poultry, providing the main source of energy (rice) and protein (chicken meat) and sustaining household food requirements. The study concludes that although smallholder cattle-owning households in Cambodia undertake a diverse range of on-farm activities, as cattle raising provides a superior income source due to higher returns and lower variable costs, it could be promoted as a preferred livelihood activity by agencies conducting rural extension activities. Although consideration of available labour and monetary funds to invest in cattle raising is required, it was observed that income from off-farm sources may prove beneficial in providing the additional monetary funds to support cattle-raising activities and assist in providing generally poor smallholder households with enhanced economic resilience.

Comparison of early detection of Fasciola hepatica in experimentally infected Merino sheep by real-time PCR, coproantigen ELISA and sedimentation.

Fasciolosis due to infection with Fasciola hepatica, Fasciola gigantica or their hybrids is a significant global cause of livestock production loss. Infection is commonly diagnosed by a labour-intensive sedimentation and faecal egg count (FEC), which has limited throughput and is only applicable after completion of the 8-12 week pre-patent period (PPP). A commercially-available ELISA for the detection of coprological antigen (coproELISA) enables detection prior to the completion of the PPP and is suitable for diagnosis of larger sample sizes, although the sensitivity reported under experimental infection settings can be difficult to replicate in the field, particularly in cattle. A recently-published real-time PCR workflow for the sensitive detection of Fasciola spp. DNA in faecal samples provides increased sample throughput, although the point at which this technique is first able to diagnose infection remains unknown. Other tools for the molecular diagnosis of fasciolosis, such as conventional PCR and loop-mediated isothermal amplification (LAMP), have been shown to detect F. hepatica DNA as early as 1 week post infection (WPI). In this study, faecal samples were collected weekly from 10 experimentally-infected Merino lambs and subjected to diagnosis via traditional sedimentation, coproELISA and real-time PCR. Samples were first considered positive at 6-8 WPI by coproELISA, real-time PCR and sedimentation, respectively. At 9 WPI 100% of samples were positive by all three methods. To evaluate the capacity of the real-time PCR approach to detect infection prior to completion of the PPP, two methods of sample preparation were compared at 2 WPI: (i) 150 mg raw faecal samples and (ii) 3 g faecal starting volume prior to sedimentation and pelleting. Neither method of sample preparation yielded positive results at 2 WPI suggesting that DNA amplification by real-time PCR is associated with faecal egg load.

Scrambled eggs: A highly sensitive molecular diagnostic workflow for Fasciola species specific detection from faecal samples.

BACKGROUND: Fasciolosis, due to Fasciola hepatica and Fasciola gigantica, is a re-emerging zoonotic parasitic disease of worldwide importance. Human and animal infections are commonly diagnosed by the traditional sedimentation and faecal egg-counting technique. However, this technique is time-consuming and prone to sensitivity errors when a large number of samples must be processed or if the operator lacks sufficient experience. Additionally, diagnosis can only be made once the 12-week pre-patent period has passed. Recently, a commercially available coprological antigen ELISA has enabled detection of F. hepatica prior to the completion of the pre-patent period, providing earlier diagnosis and increased throughput, although species differentiation is not possible in areas of parasite sympatry. Real-time PCR offers the combined benefits of highly sensitive species differentiation for medium to large sample sizes. However, no molecular diagnostic workflow currently exists for the identification of Fasciola spp. in faecal samples. METHODOLOGY/PRINCIPAL FINDINGS: A new molecular diagnostic workflow for the highly-sensitive detection and quantification of Fasciola spp. in faecal samples was developed. The technique involves sedimenting and pelleting the samples prior to DNA isolation in order to concentrate the eggs, followed by disruption by bead-beating in a benchtop homogeniser to ensure access to DNA. Although both the new molecular workflow and the traditional sedimentation technique were sensitive and specific, the new molecular workflow enabled faster sample throughput in medium to large epidemiological studies, and provided the additional benefit of speciation. Further, good correlation (R2 = 0.74-0.76) was observed between the real-time PCR values and the faecal egg count (FEC) using the new molecular workflow for all herds and sampling periods. Finally, no effect of storage in 70% ethanol was detected on sedimentation and DNA isolation outcomes; enabling transport of samples from endemic to non-endemic countries without the requirement of a complete cold chain. The commercially-available ELISA displayed poorer sensitivity, even after adjustment of the positive threshold (65-88%), compared to the sensitivity (91-100%) of the new molecular diagnostic workflow. CONCLUSIONS/SIGNIFICANCE: Species-specific assays for sensitive detection of Fasciola spp. enable ante-mortem diagnosis in both human and animal settings. This includes Southeast Asia where there are potentially many undocumented human cases and where post-mortem examination of production animals can be difficult. The new molecular workflow provides a sensitive and quantitative diagnostic approach for the rapid testing of medium to large sample sizes, potentially superseding the traditional sedimentation and FEC technique and enabling surveillance programs in locations where animal and human health funding is limited.

Improving smallholder food security through investigations of carcass composition and beef marketing of buffalo and cattle in northern Lao PDR.

This study determined the carcass composition of Lao indigenous buffalo (Bubalus bubalis) and cattle (Bos indicus), then examined trends in bovine meat marketing following review of records of beef production and prices in the two major cities of Luang Prabang (LPB) and Xieng Khoung (XK) provinces in northern Laos. Samples from 41 buffalo and 81 cattle (n = 122) were collected from animals slaughtered in May-June 2014, with live weights, carcass weights and other carcass-related variables collected. The animals were classified into four age cohort groups (<2, 2-<4, 4-6 and >6 years) with quantitative and dichotomous qualitative traits determined. There were significant differences in buffalo and cattle predicted mean carcass weights between age classification categories (p = 0.003 and 0.001) but not in dressing percentages (p = 0.1 and 0.1). The carcass weight of buffalo was 104 (±23.1)-176 (±12.0) kg compared to 65 (±8.7)-84 (±6.5) kg of cattle, with dressing percentages of 37-40 and 39-42 %, respectively. Despite an average bovine meat price increase of 42-48 % between 2011 and 2013, there was a reduction in the numbers of large ruminants slaughtered in the surveyed cities of LPB (11 %) and XK (7 %), with bovine meat availability per person of 5.2-6.6 kg (LPB) and 3.0-3.8 kg (XK). Improving the sustainability of the bovine meat supply in Laos requires a systems approach involving improvements to animal health and production, livestock marketing, plus the critical development of improved slaughterhouse facilities enabling a meat-processing sector to emerge. This development pathway is of particular importance for building the capacity of Laos to reduce food insecurity and alleviate the poverty of its largely rural smallholder community.