Plasmodium knowlesi, a parasite of long-tailed macaques (Macaca fascicularis), is an important cause of human malaria. Plasmodium cynomolgi also commonly infects these monkeys, but only one naturally acquired symptomatic human case has been reported previously.In Southeast Asia, Malariometric studies involving 5422 subjects (aged 6 months to 65 years) were conducted in 23 villages in Pailin and Battambang, western Cambodia. Parasite detection and genotyping was conducted on blood samples, using high-volume quantitative PCR (uPCR).P. cynomolgi was found in 11, P. knowlesi was found in 8, and P. vivax and P. cynomolgi were both found in 2. Only 2 subjects were female, and 14 were men aged 20–40 years. Geometric mean parasite densities were 3604 parasites/mL in P. cynomolgi infections and 52488 parasites/mL in P. knowlesi infections. All P. cynomolgi isolates had wild-type dihydrofolate reductase genes, in contrast to the very high prevalence of mutations in the human malaria parasites. Asymptomatic reappearance of P. cynomolgi occurred in 2 subjects 3 months after the first infection.Asymptomatic malaria parasite infections were detected in 1361 of 14732 samples (9.2%). Asymptomatic infections with nonhuman primate malaria parasites were found in 21 individuals living close to forested areas; P. cynomolgi and P. knowlesi infections can both occur in humans.Asymptomatic naturally acquired NCT01872702. P. knowlesi infection and 13 with P. cynomolgi infection). These parasites represented 1.9% of all identified malaria parasites.Epidemiological studies in western Cambodia identified 21 subjects with asymptomatic monkey malaria parasite infections (8 with  The excMalaysia . Laboratescribed , 3, 4, bescribed .P. knowlesi in the monkey population as a source of human infections [P. knowlesi and P. cynomolgi and present here a molecular evaluation of these infections.A malaria elimination agenda has been endorsed by all countries of the Greater Mekong Subregion in Southeast Asia . Howeverfections . Submicrfections  are impofections , 9. In tMalariometric surveys were conducted in 23 villages in the Pailin and Battambang provinces of western Cambodian as described previously , 10, 11 Plasmodium genus–specific 18S ribosomal RNA (rRNA) as described previously. This has a lower limit of accurate quantification of 22 genomes/mL [Of 6100 invited participants, 5422 provided a blood sample at least once. A total of 14732 samples were sent to the reference laboratory in Bangkok for testing by high-volume quantitative PCR (uPCR) targeting the gene encoding nomes/mL . Ethics Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and the 2 Plasmodium ovale species. A subset of samples containing Plasmodium DNA did not amplify with these species-specific probes and were investigated further.Samples testing positive by uPCR were then assessed by real-time PCR, using species-specific probes for detection of the 5 human malaria parasites: P. cynomolgi, P. knowlesi, Plasmodium coatneyi, Plasmodium brasilianum, Plasmodium inui, Plasmodium simium, Plasmodium semiovale, Plasmodium fieldi, Plasmodium fragile, Plasmodium vinckei, Plasmodium yoeli, Plasmodium chabaudi, Plasmodium berghei, and Plasmodium adleria . Two pairs of oligonucleotide primers were designed to amplify both the A and S types of the genes encoding 18S rRNA  and P. knowlesi (n = 3), we performed whole-genome amplification, using multiple displacement amplification by Phi29  according to the manufacturer’s instructions. Following collection, white blood cells were depleted from blood samples so that contaminating human DNA was less likely to limit the amplification efficiency in this system. To rule out cross-contamination during processing, 1 negative control consisting of only water was added for every 3 samples. We evaluated and compared DNA sequences and microsatellites before and after amplification in 14 samples, of which 9 were positive for P. cynomolgi, and 5 were positive for P. knowlesi. Findings were 100% concordant.For further analysis of very-low-density infections with P. cynomolgi and P. knowlesi dhfr-ts were assessed by nested PCR amplification covering the full length of dhfr-ts , followed by sequencing of the gene in an ABI Sequencer . The sequences were then aligned against the dhfr-ts of reference Cambodian strain (PCYB_053150) of P. cynomolgi and strain H (PKNH_0509600) of P. knowlesi and were analyzed using Bioedit software .Point mutations in P. cynomolgi, 8 microsatellite markers of di/tri/tetra-nucleotide repeats distributed across chromosomes 1–12, named 1.307 (TCT), 2.36 (AT), 3.574(GTGA), 4.41(TCT), 4.462(TCC), 5.956(AAG), 6.455(AGG), 7.1006(ATAC), 10.179(TA), 10.621(TGTA), 11.1096(TGTA), and 12.286(AAT), were assessed using seminested PCR as described previously [P. knowlesi, 10 microsatellite markers of trinucleotide repeats spanning chromosomes 3–13, named NC03_2(AAG), CD05_06(TAA), CD08_61(TAC), NC09_1(GAA), NC10_1(TTA), CD11_157(GAG), NC12_2(AAT), NC12_4(GAA), CD13_61(AAC), and CD13_107(AGG), were assessed using seminested PCR as described previously [To assess genetic relatedness between infecting nonhuman primate malaria parasites and between initial and recurrent infections, microsatellite genotyping was performed. For eviously . For P. eviously . The lenGenetic similarity between isolates was analyzed and a dendrogram constructed on the basis of the microsatellite markers, using an unweighted pair group method with arithmetic mean (UPGMA), performed with BioNumerics software, version 7.5 .This trial was registered on 4 June 2013 at Clinicaltrials.gov (NCT01872702).P. falciparum was detected in 114 (10.1%), P. vivax was detected in 976 (86.8%), and both species were detected in 14 (1.2%) [P. vivax species, using a less sensitive and specific real-time PCR protocol [P. vivax DNA. However, because of the 98% similarity between the genes encoding the 18S RNA of P. vivax and P. cynomolgi, which raised the possibility of misidentification, we subsequently retested the same set of samples, using the specific primate nested PCR and sequencing protocol. Three of 853 specimens (0.3%) originally identified as containing P. vivax actually contained P. cynomolgi only.Of the 14732 samples collected (7320 from Battambang and 7412 from Pailin), uPCR indicated that 1361  contained malaria parasite DNA. Of these, DNA in 1125 could be identified to the species level: d in 114 .1%, P. vprotocol , 853 samP. cynomolgi monoinfections were found in asymptomatic afebrile residents from 5 villages located close to the forest. All individuals were male; the median age was 28 years . The 5 villages comprised Chakrya, Ou Treng, Tik Mong, Veal Roleum, Phnom Rey, in Battambang, and Krong Pailin, in Pailin, Western Cambodia , and 14 were men aged 20–40 years, reflecting the demographic group most likely to enter and spend time in or near the forest.Eight dhfr-ts gene (approximately 1884 bp) was sequenced in all P. cynomolgi infections. No nonsynonymous mutations were found. Thus, 0 of the 7 drug resistance point mutations in the dhfr drug binding pocket, which are highly prevalent in P. vivax isolates in this area [P. cynomolgi contained identical repeats within the Pcdhfr of (SGDTH)(SGDNTNG)(SGDAVGT). This contrasts with P. vivax, in which the number of intragenic repeats is highly variable. In the 2 P. cynomolgi and P. vivax mixed infections, only the P. vivax dhfr-ts was amplified by PCR, presumably because P. cynomolgi was the minority population. The P. vivax dhfr-ts had the common double resistance mutations at S58R and S117N with the (GGDNTS)(GGDNADK) repeat.The full length his area , were fodhfr-ts gene (approximately 1881-bp) sequences of the 8 P. knowlesi isolates had no nonsynonymous mutations, although there was 1 deletion at codon 105 without any interruption of protein translation. There was also 1 nonsynonymous mutation (G257S) in the TS gene.The full length of P. cynomolgi isolates, 8 tandem repeat loci bearing 2–4 bp specific for P. cynomolgi were tested (n = 13). The mean number (±SE) of alleles per locus was 6.37 ± 0.29  presented with recurrent P. knowlesi parasite isolates was assessed using 7 microsatellite markers. The mean number (±SE) of alleles per locus was 2.85 ± 0.17 . P. cynomolgi in rhesus monkeys was recognized as behaving similarly to P. vivax, with frequent relapses from an exoerythrocytic source (later shown to be the dormant “hypnozoites” in the liver), and so this infection became the animal model for relapsing malaria. The potential for mosquito-to-human transmission of P. cynomolgi was illustrated first by the renowned entomologist Don Eyles when he and then his assistant became ill with malaria while studying mosquito transmission in laboratory primates [P. cynomolgi is very similar to P. vivax, although the incubation period of sporozoite-induced infections in humans is significantly longer [P. cynomolgi has not caused severe malaria in experimentally infected humans. Studies of untreated infections with both the M and B strains by Coatney et al documented persistence of the initially symptomatic P. cynomolgi human infections for as long as 58 days [P. vivax results in P. cynomolgi premunition, or vice versa, is not known. Important anopheline vectors of human malaria in Cambodia and Vietnam (Anopheles dirus and Anopheles maculatus) have been shown to carry P. cynomolgi and P. knowlesi sporozoites [P. cynomolgi infections is likely to be the highly effective forest vector A. dirus.This is the first report from a malaria-endemic area of asymptomatic infection with the nonhuman-primate malaria parasite primates . Before primates , 4. Morp 58 days , 4. Thusrozoites , 22. TheP. cynomolgi and P. vivax probably contribute to underrecognition of the monkey parasite in human infections in the Greater Mekong Subregion. Symptomatic patients in whom malaria is diagnosed either by microscopy or a genus-specific rapid diagnostic test will probably be labeled as having vivax malaria. In malariometric surveys, cross-reactivity in some of the PCR methods used may also lead to misdiagnosis, as happened early in this study. The specificity of the PCR method finally used in this study is supported by the analysis of the dhfr-ts genes. Their protein product is the target of antifolate drugs. Nonsynonymous drug resistance mutations are found in nearly all P. vivax  isolates in Cambodia and adjoining Thailand [P. cynomolgi isolates contained the corresponding Pcdhfr mutations. This is explained by the absence of drug pressure in the primate population and the likely very small contribution of human infection to P. cynomolgi transmission and epidemiology.The morphological and genetic similarities of Thailand , yet nonP. cynomolgi was recovered twice, with an intervening interval of approximately 3 months. In one case genetically distinct parasites were identified, and in the other the paired parasites were related, indicating that they were siblings derived from the same mosquito inoculum. Whether these were relapses, reinfections, or persistent infections with blood-stage parasites, with a mixed infection, cannot be determined with certainty, although reinfection is highly unlikely in the latter case. The genetic diversity of the P. cynomolgi and P. knowlesi isolates was low. The multiplicity of P. cynomolgi infections  was lower than observed in P. vivax infections from Thailand , Myanmar , and India , which may reflect the relative rarity of successful transmission to humans [P. cynomolgi human red blood cell invasion is restricted to reticulocytes expressing both transferrin receptor 1 (Trf1 or CD71) and the Duffy antigen/chemokine receptor (DARC) or CD234 [P. cynomolgi–infected individuals possess a facilitatory genetic polymorphism affecting these receptors also cannot be excluded.In 2 subjects, o humans . These sor CD234 , so the P. knowlesi infections are increasingly recognized in Southeast Asia—particularly in Malaysia [P. knowlesi is the main cause of human malaria, yet P. inui and P. cynomolgi are the more prevalent malaria parasites in long-tailed macaques, which are the main local P. knowlesi reservoir [P. cynomolgi infections have not been found there to date [P. knowlesi by the same anophelines. P. knowlesi is a quotidian parasite that may reach high densities in humans and cause potentially lethal infections, although the majority of presentations are associated with uncomplicated malaria and low-density infections [P. knowlesi may be mistaken for P. falciparum at the young ring stage and with P. malariae at the trophozoite stage. P. knowlesi is detected by pan-species rapid tests, although the limit of detection is higher than for the human malaria parasites. In early PCR protocols, there was cross-reactivity with P. vivax, its closest phylogenetic relative among the human malaria parasites, but more-recent methods such as those used here are highly specific. As with P. cynomolgi, P. knowlesi is not under antimalarial drug selection pressure and so remains fully susceptible to all of the antimalarial drugs [P. cynomolgi carried at lower parasitemia levels. Such densities, if maintained, might be too low to infect anopheline mosquitoes.Naturally acquired Malaysia , 25–30. Malaysia , 22. Inteservoir , 32. Asy to date . This maal drugs . Althougdhfr in an area where nearly all human malaria parasites are mutated at this locus provides strong supportive evidence.In malariometric surveys that employ microscopy, rapid diagnostic tests or non–species-specific PCR methods of detection, the nonhuman primate malaria parasites will not be recognized. With species-specific PCR protocols, positive samples that are not recognized by the human malaria parasite–specific probes are likely to carry nonhuman-primate malaria parasites. If there is insufficient DNA for accurate characterization, whole-genome amplification protocols may be used successfully, as in this study. If there is any uncertainty, then genes other than that encoding 18S RNA can be assessed. In this study, the finding of a “wild-type” As human malaria is controlled and the prevalence falls, primate malaria parasites will comprise an increasing proportion of cases in areas where monkeys live in close proximity to people. In this study, conducted in western Cambodia, nonhuman-primate malaria parasites comprised 1.9% of identified parasites. Regional malaria elimination assessments must recognize the propensity of these primate malaria parasites to cause asymptomatic infection, as well as symptomatic infection.The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Supplementary Table1-5Click here for additional data file.


Undernutrition is a major public health problem leading to 1 in 5 of all deaths in children under 5 years. Undernutrition leads to growth stunting and/or wasting and is often associated with environmental enteric dysfunction (EED). EED mechanisms leading to growth failure include intestinal hyperpermeability, villus blunting, malabsorption and gut inflammation. As non-invasive methods for investigating gut function in undernourished children are limited, pre-clinical models are relevant to elucidating the pathophysiological processes involved in undernutrition and EED, and to identifying novel therapeutic strategies. In many published models, undernutrition was induced using protein or micronutrient deficient diets, but these experimental models were not associated with EED. Enteropathy models mainly used gastrointestinal injury triggers. These models are presented in this review. We found only a few studies investigating the combination of undernutrition and enteropathy. This highlights the need for further developments to establish an experimental model reproducing the impact of undernutrition and enteropathy on growth, intestinal hyperpermeability and inflammation, that could be suitable for preclinical evaluation of innovative therapeutic intervention. Acute malnutrition is a major public health problem in low-income countries, affecting 51 million children under the age of five ,3. UnderAs studying gut barrier function in undernourished children is challenging, preclinical models have been developed to enable a better understanding of the mechanisms behind undernutrition and/or enteropathy. These models were also intended for the evaluation of novel therapeutics in undernutrition and/or EED. In this narrative review, we focus on available preclinical models of undernutrition and their effects on growth and gut barrier function. The respective advantages and limits of these models are discussed. A literature search using PubMed from inception to January 2019 was performed to identify relevant studies on experimental models of undernutrition and/or enteropathy. Manuscripts published in English were selected and reviewed.C. elegans exposed to starvation in an early larval stage exhibited a developmental defect [Undernutrition models are mainly induced by deficient diets, but some studies described genetic models of undernutrition. AMPK is involved in energy regulation and its genetic deletion has been investigated. For example, AMPK deficient l defect . In micel defect . These eLimitation of food provision in caloric restriction models varies from 15% to 50% in the literature . CaloricIn addition, caloric restriction might be stressful for rodents. Accordingly, in juvenile rats fed with 25% caloric restriction, plasma corticosterone levels were significantly increased . BehavioIn the first 1000 days of life, including gestation and the first two years of life, children are at risk of developing short- and long-lasting consequences for their health, including undernutrition and impaired cognitive development. Indeed, a case-control study associated mother undernutrition with an increased risk of SAM in the children . Pre-cliSalmonella and cholera vaccines [As caloric restriction leads to stress, ad libitum deficient diets may be a better alternative in order to respect the ethics for animal research. Protein-deficient diets reflect nutritional deficiencies commonly observed in undernourished children and are commonly used in the literature. While low-protein diet is isocaloric to the standard diet, several studies demonstrated increased food intake in rodents at different protein proportions, despite weight loss ,50,51. Pvaccines . By contvaccines . Fock etvaccines . Moreovevaccines . In addivaccines . Lower Tvaccines .In the LP diets previously described, mineral and vitamin proportions were adapted for rodents. In clinical settings, reduced micronutrients intake, such as vitamins and minerals, is also reduced, in addition to the low macronutrients intake in an undernourished child . For thiRegional Basic Diet (RBD) is an experimental rodent diet mimicking dietary pattern from the northeast Brazilian population, characterized by reduced protein (7%), fat (8.2%), and vitamins and minerals content, triggering clinical symptoms such as reduced weight gain in rats ,57, whicThe Lancet in 1933 and redistributed by The Bulletin of the WHO in 2003 stipulated that the origin of Kwashiorkor syndrome, characterized by bilateral edema in undernourished children, was the maize diet in Ghana, a corn-based diet [An article published in sed diet . The maised diet ,61. The sed diet . In thissed diet . The maised diet ,36.The Malawian diet (M8) is a prototypic diet based on the typical Malawian diet providing eight ingredients: corn flower, roasted peanuts, red kidney beans, pumpkin, bananas, onion tomatoes, mustard grains and water . This diE. coli (EAEC), which suggests a synergistic effect of infection and zinc deficiency in the development of enteropathy [Shigella-infected mice, a zinc-free diet prolonged Shigella gut colonization [Shigella-induced inflammation and enhanced weight recovery [Prevalence of zinc deficiency is very high in young children and infants in low-income countries . A studyeropathy . Two weeeropathy ,68. In Srecovery . Zinc derecovery . Mechanirecovery .Taking all this together, and despite the varying levels of zinc depletion among the studies, these observations suggest an impaired immune response in mice fed with zinc-deficient diets, which leads to an increased vulnerability to pathogen infection and increased virulence .Animal models of enteropathy have provided a wealth of information about gut barrier function, using microbial challenges or other gastrointestinal injury.Bacteroidetes and lower Firmicutes enrichment and unusual abundance of Prevotella and Xylanibacter genus were reported in children from rural Burkina Faso, in comparison to European children [Escherichia coli/Shigella sp. and Campylobacter sp. [Bacteroidetes and Enterobacteriaceae, in the small intestine [Firmicutes [Dysbiosis associated with child undernutrition varies from country to country , but is children . A Banglchildren . Investicter sp. . Alteratntestine ; in contrmicutes .Gnotobiotic rodents have been used as models. Indeed, transplantation of bacterial strains targeted by IgA (IgA+ bacteria consortium) from undernourished children fecal microbiota induced small intestinal and colonic epithelial disruption into gnotobiotic mice fed with Malawi-8 diet . TransplC. parvum or G. Lamblia), or bacteria.Child exposure to environmental bacteria could play a role in the development of EED, although it has not been fully demonstrated. One mechanism may be a shift in microbe-host interactions in the intestine, that might induce chronic inflammation frequently associated with undernutrition ,74,75. MCryptosporidium parvum (C. parvum), a parasitic invader of small intestine epithelial cells [C. parvum infection has a major impact on pediatric health in resource-limited countries because it increases morbidity and mortality in children [C. parvum infection might be responsible for long-lasting consequences on physical and cognitive development in children [C. parvum infection also contributes to the vicious circle of undernutrition and infection by inducing nutrients malabsorption [C. parvum infection and undernutrition in neonatal mice has been described [post-partum, 8 h on D5 and 16 h from D6 to D14) [C. parvum infection on D6 induced weight loss, with an additional effect in previously undernourished babies [C. parvum infected-undernourished babies also displayed ileal villous atrophy and crypt hypertrophy and developed higher ileal inflammation, reflected by the increased TNF-α and IFN-γ tissue levels [C. parvum oocysts infection [C parvum infection in undernourished mice increased CD8 + CD103 + T cells and B cells lamina propria infiltration [C. parvum infection exacerbated weight loss and led to decreased villous height and increased crypt depth [C. parvum infection with an LP diet (7%) in mice, for three weeks, also reported impaired gut barrier with a lower villi:crypt ratio and a lower ileal occludin protein expression, while other tight junction proteins were not affected [C. parvum-infected mice also induced intestinal inflammation, with higher fecal LCN-2 and higher MPO activity [al cells , is a maal cells ,78,79. Cchildren . A combichildren ,82. C. psorption . A modelescribed . In baby to D14) . C. parvd babies . C. parve levels . The samnfection . C parvultration . As prevpt depth ,85. A moaffected . Two weeactivity .Giardia is a non-invasive enteropathogen affecting the proximal small intestine and leading to acute watery diarrhea [Giardia infection led to a disrupted villus architecture and was associated with a higher lactulose:mannitol ratio, indicating increased intestinal permeability [Giardia Lamblia infection demonstrated decreased nutrient absorption [Giardia muris developed small intestinal injury, villus atrophy and brush border duodenal and jejunal enzymatic depletion [Giardia lamblia cultures from symptomatic and asymptomatic children inoculated in axenic Gerbils (Meriones unguiculatus) both induced growth retardation, suggesting that silent enteric dysfunction caused by the pathogen could already impair growth [Giardia infection also induced gut barrier dysfunction [Giardia lambia infection induced crypt hyperplasia without villus blunting associated with eosinophils infiltration of villus and crypt units [Giardia as an EED trigger has been debated. Observational studies in Malawi suggested links between mortality and systemic and intestinal infection in SAM children with diarrhea, but did not find an association between the intestinal presence of Giardia and mortality or diarrhea evolution [Giardia pathogens was higher in non-diarrheal stools [Giardia [Giardia infection was negatively correlated with linear growth during the first two years in the MAL-ED studies; in contrast, fecal MPO or neopterin were not increased in non-diarrheal stools [diarrhea ,88,89,90eability . These feability ,92,93. Asorption . Undernoepletion . Giardiar growth . Besidesfunction ,98. At Dpt units . Yet, thvolution . Similarl stools , while a[Giardia . Finallyl stools ,103.C. Parvum and Giardia may thus induce some features of enteropathy in models of undernutrition, which are summarized in Both Bacteroidetes and E. coli in mice. After induction, weight loss was associated with increased intestinal permeability and villus and crypt atrophy [In another model, described by Brown et al., a protein-deficient diet was combined with oral exposure to commensal  atrophy . Additio atrophy . FurtherIn undernourished children, mortality related to diarrhea is frequent . In someEscherichia coli or Salmonella enterica [Lipopolysaccharides (LPS) are a lipid-based component of the outer membrane of Gram-negative bacteria, such as enterica . LPS indenterica . LPS hasenterica . In anotenterica . The LPSenterica  and loweenterica . In a moenterica . Moreoveenterica . Intraveenterica .LPS also alters gut morphology. Indeed, intraperitoneal injection of LPS at 5 to 30 mg/kg induced duodenal villus blunting ,116. IntE. coli serotype 0111:B4) induced systemic inflammation 4 h after injection with higher IFN-γ, IL-6, MCP-1 and TNF-α levels [Escherichia coli O26:B6–1.5 mg/kg), CD1 mice displayed increased cytokines levels  [Rodent models of endotoxemia have also been used to induce systemic inflammation. Accordingly, a single i.p injection of LPS  . In summ2 which is the most abundant in the small intestine and responsible for the regulation of mucus layer production [2 production will result in a decreased mucus layer thickness [Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most frequently used medications worldwide for routine relief of pain, fever, to manage various forms of arthritis or other inflammatory disorders, and to prevent or treat cancers ,127. As oduction ; accordihickness . NSAID lhickness . Clinicahickness . A decrehickness . Thus, Nhickness ,135. Inthickness . Indomethickness ,138 in mhickness . Using ahickness . Underlyhickness . Indomethickness . In thishickness . Indomethickness . Subcutahickness  and 24 hhickness . Single hickness . Indomethickness . Moreovehickness . Subcutahickness . In a rahickness . Indomethickness . Similarhickness . In summIn the present review, we have reviewed and discussed the available animal models that may be useful to mimic the clinical features of SAM and EED. Understanding the physiological mechanisms involved during an episode of undernutrition associated with enteropathy is a critical step in developing novel therapeutic strategies. Human studies are obviously limited in terms of investigating gut function in undernourished children, in particular because of the lack of precise non-invasive methods. These limits do not apply to preclinical research. Animal models therefore offer an affordable tool to better investigate the mechanisms associated with undernutrition and enteropathy, and highlight potential biomarkers which can be translated back to the clinical condition.Although undernutrition induced by micro and/or macronutrients deficiencies can impair growth, it is not sufficient to induce enteropathy. Several combinations of infection and undernutrition can induce growth failure and affect gut barrier, but these models seem to be specific of a pathogen and may lead to uncontrolled inflammatory response, which limit their validity. Development of non-infectious models of SAM with EED by associating undernutrition with triggers that induce growth failure with moderate and prolonged enteropathy appears to be the best approach so far to set up an easy and reproducible pre-clinical model allowing for the evaluation of therapeutic strategies.


Pogostemon cablin (Blanco) Benth. (Patchouli) is not only an important essential oil plant, but also a valuable medicinal plant in China. P. cablin in China can be divided into three cultivars  and two chemotypes (pogostone-type and patchoulol-type). The pogostone-type and patchoulol-type are, respectively, used for medicinals and perfumes. In this study, we sequenced and characterized the plastid genomes for all three Chinese cultivars and aimed to develop a chemotype-specific barcode for future quality control. The plastid genomes of P. cablin cultivars ranged from 152,461 to 152,462 bp in length and comprise 114 genes including 80 protein coding genes, 30 tRNA genes, and four rRNA genes. Phylogenetic analyses suggested that P. cablin cultivars clustered with the other two Pogostemon species with strong support. Although extremely conserved in P. cablin plastid genomes, 58 cpSSRs were filtered out among the three cultivars. One single variable locus, cpSSR, was discovered. The cpSSR genotypes successfully matched the chemotypes of Chinese patchouli, which was further supported by PCR-based Sanger sequences in more Chinese patchouli samples. The barcode developed in this study is thought to be a simple and reliable quality control method for Chinese P. cablin on the market. Pogostemon cablin (Blanco) Benth. (Lamiaceae), commonly called Patchouli, is a commercially important plant for its essential oil (patchouli oil). The species has been cultivated widely in China, India, Indonesia, Malaysia, the Philippines, and Singapore [P. cablin is lacking, and its wild populations may be extinct [P. cablin is reported as one of the top 20 essential oil yielding plants and is considered to have tremendous economic potential [ingapore , 2. Howe extinct –5. Patch extinct , 6. P. cotential .P. cablin in China is also an important traditional Chinese materia medica for dispelling dampness in the middle-energizer, summer heat and dampness, acedia, fullness in the chest, hypochondrium issues, cramps, diarrhea, and so on [P. cablin have evolved diverse morphological characteristics and traits , Pogostemon cablin ‘Gaoyao’ , and Pogostemon cablin ‘Hainan’  [P. cablin populations from Zhanjiang (Guangdong Province) were formerly treated as P. cablin ‘Zhanjiang’ , anrovince) , 12. Thehe 1960s , 12, thuhe 1960s .P. cablin [P. cablin [P. cablin cultivars exhibit significant differences in quality and bioactive components, as these factors are influenced by climate, soil nutrients, and water in the different locations [P. cablin in China can be divided into two chemotypes: pogostone-type, with a high content of pogostone and a low content of patchouli alcohol, and patchoulol-type, which has a high content of patchouli alcohol but a low content of pogostone [More than 140 compounds have been isolated and identified from . cablin , 15. Difocations . Based oogostone , 17, 18.ogostone , 15, 18.However, the production of pogostone, likely the main effective compound in medicinals, is extremely low, since cultivars Shipai and Gaoyao of the chemotype can be only cultivated in the suburbs of Guangzhou (Guangdong) and Gaoyao , respectively. The cultivation of the authentic herb of Shipai has been severely impacted with the urban area expansion of Guangzhou City , and itsP. cablin in the commercial market. An easy and reliable way to identify the two chemotypes of P. cablin is crucial but has not yet been developed. Previous studies have shown the potential use of Gas chromatography-mass spectrometry (GC-MS) fingerprints of Chinese P. cablin  for quality control [matK and nuclear 18S rRNA could be used to distinguish the two chemotypes, but these markers failed when they were re-sequenced and analyzed more recently by Yao et al. [It is morphologically difficult to distinguish the two chemotypes of Chinese  control , 19, 20, control  indicateo et al. .Plastid genomes have been recently recommended as important “extended barcodes” , 22. BasP. cablin cultivars representing two chemotypes (pogostone-type and patchoulol-type). We aimed to characterize all three plastid genomes to develop specific barcodes for discriminating the two chemotypes of Chinese P. cablin. The main objective of this study was to provide an accurate method for quality control of the medicinal plants and plant medicines on the market.In this study, we sequenced and assembled the plastid genomes of three Chinese The locations of the field studies are neither private lands nor protected areas. No specific permissions were required for the corresponding locations/activities.Pogostemon cablin ‘Shipai’), Gaoyao (Pogostemon cablin ‘Gaoyao’), and Hainan (Pogostemon cablin ‘Hainan’) [P. cablin.In the present study, we collected all three Chinese patchouli cultivars, Shipai (Hainan’) . For theThe accessions were obtained from the South China Botanical Garden and the Guangdong Institute of Chinese Materia Medica, China. Total genomic DNA was extracted from young leaves using a modified cetyltrimethylammonium bromide (CTAB) method .The plastid genome of the accession from the Shipai cultivar was recovered by long-range PCR enrichment using nine conserved primers . The genhttp://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used to assess the quality of raw reads, and Trimmomatic [P. cablin plastid genomes were initially assembled using SPAdes v3.10.1 [Pogostemon yatabeanus (Makino) Press (KP718618) using the Basic Local Alignment Search Tool (BLAST) . According to the reference genome sequence (KP718618), the order and direction of the aligned contigs were determined. Aligned contigs were subsequently manually assembled to construct a preliminary sequence of the P. cablin plastome.FastQC 0.11.5  using Geneious R9 v9.1.4 . These transferred gene annotations were manually corrected by the translation results and comparisons were made to homologous genes from other sequenced plastid genomes in Lamiaceae. The tRNA genes were verified using ARAGORN [http://ogdraw.mpimp-golm.mpg.de/) [The resulting assembly genome sequence was used as a reference, to which initial paired-end plastid reads were mapped using Bowtie 2.3.1 . Finally ARAGORN , with nempg.de/) .Pogostemon. We then calculated the percentage of nucleotide variability for each molecular region by dividing the numbers of nucleotide substitutions (or indels) by the number of nucleotides of the aligned sequence length.The protein-coding genes (PCGs) and non-coding regions (intergenic spacers and introns) were extracted and aligned with MAFFT , then thP. cablin plastid genomes in this study), and were aligned using MAFFT [P. cablin using RAxML-HPC v8 [According to the latest molecular results on Lamiaceae published by Li et al. , 14 plasng MAFFT . A phyloL-HPC v8  with theP. cablin and its relatives (P. yatabeanus and P. stellatus) were detected using MISA [Simple sequence repeats (SSRs) in plastid genomes of ing MISA  with theP. cablin cultivars were aligned by MAFFT [P. cablin, we tested the barcodes in all accessions by Sanger sequencing. Primer pairs were then designed using Primer3 software [oC, and other settings were maintained at default values. A 25 μL polymerase chain reaction (PCR) reaction mixture was prepared and amplified according to the procedure described by Zhang et al. [P. cablin on 2% agarose gels. Amplicons with single, clear bands on agarose gels were purified and sequenced in both directions on an ABI3730X sequencer  using the amplification primers. The sequenced genotype for each chemotype was deposited in GenBank , and c. 2 GB paired-end reads of accession of cultivar Shipai using the Miseq platform. The sizes of the assembled P. cablin plastid genome are 152,461 bp for Shipai and Gaoyao cultivars, and 152,462 bp for the Hainan cultivar. The complete plastid genomes of P. cablin with annotations have been submitted to GenBank . All plastid genomes of P. cablin have the typical quadripartite structure of angiosperm plastid genomes, with a pair of IRa and IRb of 25,662 bp for all cultivars, LSC of 83,553 bp for Shipai and Gaoyao and 83,554 bp for Hainan, and SSC of 17,584 bp for all cultivars , seven tRNAs , and four rRNAs  were duplicated in the IRs . ComparNA genes . The twoPogostemon were carefully examined in this study. In total, we identified 21 introns and 100 intergenic spacers. Nine protein-coding genes and six tRNAs contained the single intron, while three protein-coding genes possessed two introns  by the number of nucleotides of the aligned sequence length. Specifically, we termed S/P for the percentage of Single Nucleotide Polymorphisms (SNPs) for each molecular region, and I/P for the percentage of Indels  to 0.044 (rpl16), and was 0.014 on average . Considering the limitation of Sanger sequencing, fragments 500–1,500 bp long is most suitable for Sanger sequencing. We therefore set a strategy for molecular marker selection, of which fragment sizes should range from 500 to 1,500 bp, and the percentage variabilities should be larger than average. Twenty-five highly variable regions, consisting of 14 noncoding regions and 11 coding regions, were summarized in Pogostemon species, but additional studies are required in the future to confirm this.The percentage variability of coding regions (introns in coding regions were included herein) were estimated as above. Among the SNPs of coding regions, the percentage variability ranged from 0  are needed to construct a robust phylogeny and evolutionary history of Pogostemon [Our phylogenetic trees based on different datasets  exhibited congruent topologies in  species . As expe support . Though gostemon , more Pogostemon .Pogostemon phylogeny in the future.For over two decades, a driving question in the theory of phylogenetic experimental design has been how to select a set of characters that are evolving at rates appropriate for resolving a given phylogenetic problem . Among tThough the rate of molecular evolution in the plastid genome is relatively slow, noncoding plastid DNA can provide informative variation at the species and population level . BecausePogostemon cablin plastid genomes, including 58 mononucleotides, but no other repeats were found (rpoC2 (×2), atpB, and ycf1 (×2)), and eleven are located in seven introns , rpoC1, ycf3, petB (×3), and rpl16 (×2)) of the P. cablin plastid genomes. Most of these SSR loci are found in the LSC region (84.48%), followed by the SSC (12.07%) and IR regions (3.44%). We also listed the cpSSRs of P. yatabeanus and P. stellatus in Pogostemon species, and only one and two dinucleotide repeats, respectively, were found in P. yatabeanus and P. stellatus. These cpSSRs of all three Pogostemon plastid genomes are generally A or T repeats, which is consistent with the AT-richness mainly in intergenic and intron regions of plant plastid genomes [A total of 58 cpSSRs with lengths of at least 10 bp were detected throughout the re found . These c genomes .P. cablin. The low genetic diversity has been verified by genome scanning with specific-locus amplified fragment sequencing (SLAF-seq) [P. cablin is supported by this study, since no nucleotide polymorphisms and indels were found among the plastid genomes of all three Chinese cultivars, except one cpSSR locus. The cpSSR locus is located in the intergenic region between ycf3 and trnS-GGA  bp), and it exhibited a variation in A/T repeats among the P. cablin cultivars. It is invaluable that the patchoulol-type (Hainan) showed (A/T)11 (which indicates Genotype A herein) in the locus, while the pogostone-type (Shipai and Gaoyao) exhibited (A/T)10 (Genotype B herein). Using high-fidelity PCR enzymes , we amplified and sequenced the molecular regions (ranging from 967 bp to 968 bp) throughout all accessions using primer pairs . The scaled map of the locus with primer positions is illustrated in P. cablin.Since it seldom flowers , extensiLAF-seq) .The prevmatK and nuclear 18S rRNA. However, matK and ITS  did not have any mutations corresponding to the chemotypes Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.S3 Appendix(DOCX)Click here for additional data file.S4 Appendix(A) CDS; (B) intergenic spacers; (C) intron; (D) LSC; (E) SSC; (F) IR.(PDF)Click here for additional data file.S5 Appendix(A) CDS; (B) intergenic spacers; (C) intron; (D) IR; (E) LSC; (F) SSC; (G) completed plastid genomes.(PDF)Click here for additional data file.


Bifidobacterium in health-promoting gut bacteria in humans, little is known about their functions in wild animals, especially non-human primates. It is difficult to determine in vivo the function of Bifidobacterium in wild animals due to the limited accessibility of studying target animals in natural conditions. However, the genomic characteristics of Bifidobacterium obtained from the feces of wild animals can provide insight into their functionality in the gut. Here, we analyzed the whole genomes of 12 B. moukalabense strains isolated from seven feces samples of wild western lowland gorillas (Gorilla gorilla gorilla), three samples of wild central chimpanzees (Pan troglodytes troglodytes) and two samples of wild forest elephants (Loxodonta cyclotis) in Moukalaba-Doudou National Park, Gabon. In addition, we analyzed the fecal bacterial communities of six wild western lowland gorillas by meta 16S rRNA gene analyses with next generation sequencing. Although the abundance of the genus Bifidobacterium was as low as 0.2% in the total reads, a whole genome analysis of B. moukalabense suggested its contribution digestion of food and nutrition of frugivore/folivore animals. Specifically, the whole genome analysis indicated the involvement of B. moukalabense in hemicellulose degradation for short chain fatty acid production and nucleic acid utilization as nitrogen resources. In comparison with human-associated Bifidobacterium spp., genes for carbohydrate transport and metabolism are not conserved in these wild species. In particular the glycosidases, which are found in all 12 strains of B. moukalabense, were variably detected, or not detected, in human-associated species.Despite the essential role of  Bifidobacterium is the unique Actinobacteria, which is adapted to animal intestinal tracts. Bifidobacteria have been commonly recognized as health-promoting gut bacteria in many health-oriented research studies [Bifidobacterium comprises 70 established species with 10 subspecies according to LPSN (http://www.bacterio.net/bifidobacterium.html). Within these 70 species, human-associated species of Bifidobacterium are abundant. In contrast, other animal species often carry bifidobacteria with much lower levels of diversity [Bifidobacterium as one of the most abundant bacterial genera (~10%) [B. adolescentis-group, B. longum-group or B. scardovi-group and B. bifidum have been recognized as human-associated bifidobacteria [The gut microbiome is composed of an immense diversity of microorganisms , and rec studies ,10,11,12iversity . In facta (~10%) , and to bacteria . The widbacteria .Bifidobacterium have been conducted to assess the genomic characteristics of this particular genus in terms of adaptation to the human intestinal tract [Bifidobacterium spp. has been identified in some of the probiotic strains [Bifidobacterium spp. may be related.Several comparative genomic studies of al tract ,18,19. T strains ,21,22. TB. moukalabense was first isolated from a wild western lowland gorilla (Gorilla gorilla gorilla) [B. moukalabense belongs to the B. adolescentis-group and is closely related to B. catenulatum and B. pseudocatenulatum. The draft genome sequence reported later on this strain GG01T indicated that this strain is close to B. dentium, the other member of the B. adolescentis group [B. moukalabense and those from human associated Bifidobacterium can address the question of why this species has not been identified from human microbiota, while it was identified in Homininae primates.gorilla) . Accordiis group . GeneticB. moukalabense [B. moukalabense from wild western lowland gorillas, wild central chimpanzees (Pan troglodytes troglodytes) and wild forest elephants (Loxodonta cyclotis) in the same forest in Moukalaba-Doudou National Park, Gabon. In this study, we carried out genomic analyses of 12 strains involving GG01T to reveal the genomic characteristics by comparing the annotated gene functionality with those of human-associated Bifidobacterium species. The genomic differences and similarities between B. moukalabense and human-associated Bifidobacterium spp. suggested functional adaptations of B. moukalabense to the frugivore/folivore feeding behavior of the host.Since our first isolation of this particular alabense , we haveBifidobacteria were isolated from the feces of wild western lowland gorillas, central chimpanzees and forest elephants in Moukalaba-Doudou National Park in Gabon (v/v) Triton X-100 [in Gabon . Seven gon X-100 .Taq polymerase  and primers 27F and 1492R [PCR amplification of the 16S rRNA gene was performed using EX-nd 1492R  with a tAn aliquot of DNA (40–80 ng) from each isolate was sheared to a target peak size of 550 bp using the Covaris S220 Focused-Ultrasonicator system  according to the manufacturer’s recommendations. To generate DNA sequencing libraries for high-throughput DNA sequencing, a TruSeq DNA PCR-Free Library Preparation Kit  was used according to the manufacturer’s instructions. The library products were isolated in agars gels (size of 500–800 bp) and purified by use of NucleoSpin Gel and PCR Clean-up . The sequencing library was used as a template for paired-end sequencing using a MiSeq Reagent Kit v3 (600 cyc) and the MiSeq desktop sequencer . Read files (fastq.gz) sequence reads were generated  using MiSeq Reporter software version 2.3.32 .B. moukalabense group from all isolates with human-associated Bifidobacterium, i.e., B. catenulatum, B. dentium, B. pseudocatenulatum, and B. longum, to reveal potentially adaptive function of Bifidobacterium in non-human frugivore/folivore animals. B. longum, which constitutes a neighboring phylogenetic group of the B. adolescentis group [−5 and a minimum coverage of 75% in the BLASTP alignments. The core genome was constructed using clusters of only single-copy genes from all sequenced and reference strains, and a pangenome matrix was constructed to manually inspect the non-core homologues. B. catenulatum was used as the reference for annotation for all homologous clusters in the core genome set, while clusters outside the core genome  were manually curated by inspecting the annotations for all strains present in the cluster. The proteins were classified by the COG functional category [B. moukalabense, B. denitium, B. catenulatum and B. pseudocatenulatum were reconstructed based on their genomic data with the maximum likelihood method and the Bayesian method. The nucleotide substitution models were evaluated by the criterion of AIC, AICc, and BIC with the ModelFinder, and the GTR+I+Γ model was selected as the best model in all criterion. Maximum likelihood tree was reconstructed using RAxML version 8.2.10 [−18.We discarded the Illumina MiSeq platform reads that contained ambiguous nucleotides, low qualily fastq reads (< average QV 25) and those mapped to the PhiX genome sequence. We also removed Illumina adapter sequences. These filtration steps were performed using Prinseq version 0.20.4 , Bowtie is group  was invois group  with a mcategory . The genn 8.2.10  with then 8.2.10  with 100n 8.2.10  using HMn 8.2.10  with a mBifidobacterium genomes, derived from 9428 human metagenome samples, were obtained from a database (http://opendata.lifebit.ai/table/SGB) [Bifidobacterium genomes were predicted using Prodigal version 2.6.3 with -p meta parameter [B. moukalabense were searched for against the 6,105,571 amino acid sequences using MMseqs2 with -s 4 parameter [The 3973 draft ble/SGB) . Proteinarameter . The 583Feces samples from gorillas were collected in August 2011 in Moukaraba-Doudou National Park, as indicated elsewhere . BrieflyThe feces of wild gorillas, chimpanzees, and elephants were collected non-invasively, as indicated elsewhere . The samB. moukalabense were 2.5 Mb ± 0.59 (mean ± SD) with nearly 59.9% GC content. These genomes harbor from 1919 to 2143 coding sequences (CDSs), from 57 to 64 rRNAs, and 4 or 5 tRNAs. No plasmid associated genes were detected.The draft genome sequences of the 12 strains included 18 to 33 contigs with varying size up to 2.40–2.59 Mbps  with relB. moukalabense was confirmed to be phylogenetically close to B. catenulatum and B. pseudocatenulatum and is a monophyletic species belonging to the B. adolescentis-group by 16S rRNA gene analysis  and 3973 meta-bifidobacterial genomes reconstructed from a human metagenome database [B. moukalabense genomes (PRJDB7909 and AZMV00000000) and seven human-associated Bifidobacterium.The total number of proteins in this study was 39,774. They were organized into 5832 clusters, hereafter called “orthologous clusters”. We categorized these proteins and orthologous clusters into seven levels according to the conservation level when compared with human-associated . longum . The fredatabase , the conB. moukalabense and human-associated species: defense mechanisms (V), mobilome (X), and secondary metabolites metabolism (Q). The category of transcription (K), cell wall and membrane (M) and carbohydrate transport and metabolism (G) are also the less conservative.The conservation level, broken down by the COG category, is shown in B. moukalabense, those in conservation levels 1 and 2 that are present in all B. moukalabense strains, and were variably present or not present in the human-associated species, were particularly over-represented in this functional category, carbohydrate transport and metabolism, showing that the genes involved in the carbohydrate metabolism are heterogeneous in the human-associated species  in Although the proteins associated with amino acid metabolism (E) and nucleotide metabolism (F) are conservative , a clearBifidobacterium in fecal microbiota was calculated to be as low as 0.24% ± 0.15 (mean ± SD) in wild western lowland gorillas  in Bossou, Guinea and B. moukalabense is the sole Bifidobacterium isolated from wild western lowland gorillas [Bifidobacterium isolated from wild central chimpanzees in this study. However, only low levels (0.24% abundance) of the genus Bifidobacterium were recorded for six gorilla feces samples taken from the Moukalaba-Doudou National Park. Like wild western lowland gorillas, wild central chimpanzees showed low levels of Bifidobacterium in their gut microbiota [B. moukalabense was also isolated from wild forest elephants in the same forest, although low levels of Bifidobacterium in their gut were reported [Bifidobacterium by western lowland gorillas, central chimpanzees and forest elephants suggests that B. moukalabense is associated with frugivore/folivore feeding.In contrast to humans, gorillas ,45. Thiscrobiota . It is nreported ,47. Sincreported ,49,50, tB. moukalabense belong to the B. adolescentis group which comprises B. dentium, B. catenulatum and B. pseudocatenulatum associated with the human gut. Captive-bred gorillas and chimpanzees harbored B. dentium, B. catenulatum and B. pseudocatenulatum instead of B. angulatum or B. moukalabense [Bifidobacterium with such high density and divergency as in humans, as suggested by the present study , they can represent the human gut microbiome under a wide variety of feeding conditions.Other strongly biased distributions of genes concerning nitrogen-scavenging capacity, such as nucleic acid catabolism, may also reflect the frugivore/folivore food habit of the host, in which the limitation of protein, i.e., amino acid, availability in the gut is expected . Similarisolates . Insteadisolates . Wild boisolates . Similar protein . The afoB. moukalabense is a unique Bifidobacterium which is specialized to frugivore/folivore feeding habits of its host. In this context, this Bifidobacterium can reside in the gut of the typical herbivore animal like African forest elephants. But adaptation to frugivore/folivore feeding habits may not have been advantageous in omnivore human ancestors, who developed carnivore food habits [In conclusion, d habits .


Ticks and tick-borne pathogens (TTBP) are a major constraint to livestock production in Pakistan; despite a high prevalence of TTBPs, knowledge on the capacity of Pakistani ticks to carry pathogens and endosymbionts is limited. Furthermore, mixed infections with multiple microorganisms further complicate and limit the detection potential of traditional diagnostic methods. The present study investigated the tick-borne microorganisms in bovine ticks in Pakistan, employing a high-throughput microfluidic real-time PCR based technique.n = 116) and water buffaloes (n = 88) from 30 villages across six districts located in five agro-ecological zones (AEZs) of Pakistan from September to November 2017. The microfluidic real-time PCR was used to test the genomic DNA of individual ticks for the presence of 27 bacterial and eight parasitic microorganisms. Phylogenetic methods were used to assess the genetic relationship of DNA sequences determined herein.Ticks were collected from clinically healthy cattle (Babesia/Theileria spp.) were the most prevalent , followed by Ehrlichia spp.  and Anaplasma marginale . Anaplasma phagocytophilum, A. ovis, A. centrale, Babesia ovis, Borrelia spp., Rickettsia spp., R. massiliae, Bartonella spp. and Hepatozoon spp. were also detected. Endosymbionts such as Francisella-like  and Coxiella-like  organisms were also detected in ticks. The highest diversity of microorganisms was detected in Hyalomma anatolicum ticks (test-positive for 14/14 microorganisms), followed by Rhipicephalus microplus (4/14), Hy. hussaini (3/14) and Rh. annulatus (2/14). Ticks collected from cattle carried significantly more frequently piroplasms  than those from buffaloes . However, the overall prevalence of microorganisms did not vary significantly among ticks from the two host species as well as across different AEZs.PCR detected DNA of at least one microorganism in each of 221 ticks tested . DNA-based detection inferred that single pathogens/endosymbionts were the most common  followed by double , triple , quadruple  and quintuple  mixed infections. Piroplasms (To our knowledge, this is the first study to investigate a wide range of tick-borne microorganisms in bovine ticks using a high-throughput diagnostic method from different AEZs in Pakistan. These findings will aid in establishing the distribution patterns and the control of tick-borne pathogens of bovines in Pakistan. Ticks (Acari: Ixodida) are obligate, blood-sucking ectoparasites of vertebrates and are distributed worldwide . They poBubalus bubalis; n = 40 million) and cattle  .Tick specimens from each animal were stored in separate tubes containing 70% ethanol. Subsequently, each tick was morphologically characterized under a dissecting microscope  using dichotomous keys , 43. FolFor DNA amplification, the Perfecta Preamp Supermix  was used according to the manufacturer’s guidelines. Primers  were pooled combining equal volumes , and the reaction was performed in a final volume of 5 μl containing 1 μl Perfecta Preamp 5×, 1.25 μl pooled primers mix, 1.5 μl distilled water and 1.25 μl DNA. PCR cycling conditions were one cycle at 95 °C for 2 min followed by 14 cycles at 95 °C for 10 s and 60 °C for 3 min. At the end of the cycling program, the reactions were diluted as 1:10 and the amplicons were stored at – 20 °C until further use.TM real-time PCR system . These chips dispensed 48 samples and 48 PCR mixes into individual wells, followed by on-chip real-time PCR reactions in individual chambers and thermal cycling, resulting in 2,304 individual reactions. For more details regarding the development of this high-throughput tool based on real-time microfluidic PCRs , please see Michelet et al. 2014 [High-throughput microfluidic amplification was performed for major TBPs and potential endosymbionts using 48.48 dynamics array in a Bio-Markal. 2014 .Borrelia spp. (23S), Bo. burgdorferi (rpoB), Bo. garinii (rpoB), Bo. afzelii (fla), Bo. valaisiana (ospA), Bo. lusitaniae (rpoB), Bo. spielmanii (fla), Bo. bissettii (rpoB), Bo. miyamotoi (glpQ), Bo. mayonii (fla), Bo. bavariensis (pyrG), Anaplasma spp. (16S), A. marginale (msp1), A. platys (groEL), A. phagocytophilum (msp2), A. ovis (msp4), A. centrale (groEL), A. bovis (groEL), Ehrlichia spp. (16S), E. canis (gltA), Neorickettsia mikurensis (groEL), Rickettsia spp. (gltA), R. conorii (ITS), R. slovaca (ITS), R. massiliae (ITS), R. helvetica (ITS), R. aeschlimannii (ITS), R. felis (orfB), Bartonella spp. (ssrA), Ba. henselae (pap31), Francisella spp. (tul4 and fopA), Coxiella spp. (IS1111 and icd), Babesia microti (CCTeta), B. canis (18S), B. ovis (18S), B. bovis (CCTeta), B. caballi (rap1), Babesia str. EU1 (18S), B. divergens (hsp70), B. vulpes (cox1), Theileria spp. (18S) and Hepatozoon spp. (18S). Briefly, amplifications were performed using 6-carboxyfluorescein (FAM)- and black hole quencher (BHQ1)-labelled TaqMan probes with TaqMan Gene expression master mix as per manufacturer’s recommendations  [16S gene served as a positive control for the confirmation of DNA extraction. To assess PCR inhibitory molecules present in tick DNA samples, DNA from Escherichia coli (EDL933 strain) was added to each sample as an internal inhibition control, and primers and probe specific for the eae gene of E. coli were used.Targeted microorganisms (and markers) were ts, USA) . PCR cycTM system. Amplicons were sequenced by Eurofins MWG Operon  and assembled using the Geneious Prime software . An online BLAST  was used to identify the sequenced organisms.Microfluidic real-time PCR results were confirmed through conventional and nested PCR using different primers  genes were used to assess the genetic relationships with those of species of Babesia/Theileria, Ehrlichia and Rickettsia, respectively. Reference sequences were downloaded for each pathogen from GenBank and aligned separately  with sequences obtained in this study, using ClustalW [th tree was saved (samplefreq = 400). For the NJ tree estimations, evolutionary distances were computed using the p-distance method whereas for the ML method, initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log-likelihood value. All positions containing gaps and missing data were eliminated. Bootstrapping method  was used to assess the reliability of internal branches and all trees were visualized and edited using MEGA. Plasmodium falciparum (GenBank: M19172), A. marginale (GenBank: AF414872) and R. bellii (GenBank: AY362703) were used as outgroups for Babesia/Theileria, Ehrlichia and Rickettsia, respectively.The partial nucleotide sequences of microorganisms obtained for ClustalW  in the sClustalW . The besClustalW . Each BaChi-square and Fisher’s exact tests were used to evaluate the overall prevalence of microorganisms and to compare their prevalence in different AEZs. Data were analyzed using GraphPad 5 Prism program . The multiple correspondence analysis (MCA) was used to analyze the pattern of the association between infections (co-infections) and their distribution across provinces, districts and bovine host species. The inertia values were calculated by standard ‘Burt matrix’ method. The analyses were performed using the software, Statgraphics Centurion v. 16.1.03 .Francisella-like endosymbionts (FLEs) were the most common  microorganisms detected in ticks , A. marginale , Borrelia spp. , A. centrale , Rickettsia spp. , A. ovis , R. massiliae  and Coxiella-like endosymbionts (CLEs) . Furthermore, A. phagocytophilum, Bartonella spp., Babesia ovis and Hepatozoon spp. were also detected in 0.4% of ticks.PCR detected DNA of at least one microorganism in each of 221 ticks tested   across districts  in different AEZs . The highest diversity of microorganisms was found in district Okara where DNA of eight of 14 (57.1%) microorganisms tested for was detected followed by Bahawalpur and Sukkur (7/14) and Layyah and Thatta (6/14) , Jhelum and Thatta (P = 0.0010), Jhelum and Sukkur (P = 0.0017) Jhelum and Layyah (P = 0.0214) and Okara and Jhelum (P = 0.0027). At the provincial level, the diversity of microorganisms in ticks was higher in Punjab (11/14) than in Sindh (9/14) (Table P = 0.0011) than buffalo ticks ; however, the overall prevalence of microorganisms in ticks did not vary significantly between both host species  of them being test-positive for DNA of at least one microorganism, followed by Rh. microplus (4/14), Hy. hussaini (3/14) and Rh. annulatus (2/14)  was found in hussaini /14 and REhrlichia spp.  whereas the most common triple co-infection was due to FLEs, piroplasm species and Ehrlichia spp.   while the remaining tick species were positive for up to triple co-infections (Table Single infections with DNA of various microorganisms were present in 41% 96/234) of ticks, with FLEs  and piroplasm spp.  being the main microorganisms detected  detected herein grouped with those previously published sequences from Thailand (GenBank:AF497581), Tibet, China (GenBank:AF414399) and Multan, Pakistan (GenBank:MH250197)  were identified and one of these grouped with T. annulata sequences from Pakistan (GenBank: JQ743630) and Turkey (GenBank: MK918607), whereas the second sequence clustered with that of B. occultans from South Africa (GenBank: U09834)  was found which grouped with R. aeschlimannii (GenBank: DQ235776) from Russia, R. rhipicephali (GenBank: U59721) from USA and Rickettsia sp. Bar (GenBank: U59720) from Spain  were used to analyze (separately) genetic relationships of in Figs. , 5, 6. TMultiple correspondence analysis revealed no correlation between microorganisms and different districts, hosts or AEZs as microorganism clustered close to the centre of multidimensional matrices  and 12 bacteria  of veterinary and/or public health significance. Interestingly, this study revealed that bovine ticks from Pakistan have (i) a diverse range of endosymbionts, with a higher prevalence of FLEs (91.5%); (ii) a high prevalence of TBPs (127 positive of 234 ticks tested); and (iii) a high frequency of co-infections . Due to low cycle threshold (Cq) values, genetic characterization was not successful for some of the detected pathogens. As feeding ticks were collected from bovine hosts, we could not justify the bovine or tick origin of detected microorganisms. Likewise, this study does not argue the co-transmission of multiple microorganisms from a tick to the bovine host. However, such studies provide a snapshot of potential TBPs present in a region and help understanding disease(s) dynamics. Furthermore, all ticks tested in this study were collected during one season of the year, and therefore, this study does not inform about the seasonal variation of TBPs.This is the first study to utilize a high-throughput microfluidic technique for the detection of TBPs in bovine ticks collected from five AEZs of Pakistan. This real-time PCR technique offers a unique ability to detect multiple pathogens in ticks –39; hereAnaplasma spp.  in three species of ticks, Hy. anatolicum, Rh. microplus and Rh. annulatus. Bovine anaplasmosis is an endemic TBD in Pakistan, and is mainly caused by A. marginale [A. marginale in ticks from Okara district which could be attributed to high prevalence of Rh. microplus in this region. Furthermore, this tick is the main vector for A. marginale [A. phagocytophilum DNA in Hy. anatolicum for the first time from Pakistan. Anaplasma phagocytophilum is a zoonotic TBP infecting several mammalian species and can cause an acute febrile condition in humans, known as human granulocytic anaplasmosis [A. phagocytophilum is mainly responsible for equine granulocytic anaplasmosis and has been reported from equines [Anaplasma species has been reported in bovines from Pakistan [A. phagocytophilum are Ixodes spp. but several other tick species have also been found positive for its DNA [A. ovis was detected in four specimens of Hy. anatolicum and it causes ovine anaplasmosis [A. ovis has been detected in small ruminants from Khyber Pakhtunkhwa [Hy. anatolicum, Hy. dromedarii and Rh. microplus) from Punjab, Pakistan [A. ovis DNA in multiple tick species indicates the challenges associated with multi-species livestock farming by small-holder dairy farmers.In this study, we detected the DNA of four arginale , 21, 22.arginale . This stlasmosis . In Paki equines . RecentlPakistan . Main ti its DNA . Additiolasmosis –54. Prevhtunkhwa  and in tPakistan . The detEhrlichia species in 47 specimens of Hy. anatolicum from all six districts, with the highest percentage in Layyah (11/26) and lowest in Jhelum (1/24). Both districts are located within the same AEZ but hold a diverse topography because Jhelum district has plain and mountainous areas while Layyah district is comprised of desert and plain lands. Furthermore, in Layyah, summer temperatures are higher, and the livestock population there is three times larger than in Jhelum, thereby possibly providing more suitable host and environmental factors for the growth of ticks and the transmission of TBPs. Sequence and phylogenetic analyses of 16S rRNA gene fragments of Ehrlichia spp. detected in this study showed a close similarity to those previously published from China  [Ehrlichia spp. in ticks reported in our study as well as in the previous study [Hyalomma ticks might be transmitting ehrlichiosis in bovines in Pakistan; however, this hypothesis warrants further investigation.We found the DNA of . Tibet; ), Pakist Multan; ) and Tha. EBm52; )  . The higus study  suggestsTheileria/Babesia spp.; 31.6%) were detected in three tick species . Microfluidic results were validated using piroplasm-specific primers targeting 18S rRNA gene and revealed that out of seven Theileria/Babesia-positive samples sequenced, five identical sequences belonged to T. annulata while two remaining identical sequences were identified as B. occultans. Since sequencing was not undertaken for all samples, we were not able to determine individual prevalence rates for Babesia and Theileria spp. in ticks. Babesia ovis was also found in one tick sample using species-specific primers. All of these pathogens have been previously reported from Pakistan [T. annulata and is responsible for high economic losses due to reduced production and mortalities, particularly in exotic and cross-bred animals [T. orientalis has been reported in bovines and ticks from Pakistan [Theileria species complex still remain to be seen. Bovine babesiosis is another important TBD in Pakistan and is mainly caused by B. bovis and B. bigemina [B. bovis DNA in any tick sample using species-specific primers and no tick was tested for B. bigemina, both of which were previously reported to occur in Pakistan [Babesia/Theileria genera-specific primers, B. occultans was found in two Hy. anatolicum samples. Since its first detection in South Africa in 1981, B. occultans had been considered as apathogenic with its distribution limited to sub-Saharan countries [B. occultans might become an important TBP for bovines in future. Furthermore, we found that ticks from cattle carried significantly higher piroplasms compared to those collected buffaloes which might be due to the natural variation in the susceptibility of the hosts to different pathogens as buffaloes are known to be asymptomatic reservoirs of Babesia spp., which are pathogenic to cattle [A high number of piroplasms  and Rh. microplus (n = 1) in Pakistan. However, none of the seven species-specific primer pairs employed in this study could verify species identity, and therefore, the pathogenic and/or zoonotic potential of the detected Borrelia species could not be established. Borrelia species include spirochetes belonging to Lyme borreliosis and relapsing fever spirochete groups as well as intermittent clades and are transmitted by the body louse and hard and soft ticks [Borrelia spp. and to assess their pathogenic and zoonotic potential.This study detected, for the first time, DNA of ft ticks , 70. TheHy. hussaini and three Hy. anatolicum ticks contained DNA of R. massiliae, whereas, five ticks (one Hy. hussaini and four Hy. anatolicum) were positive for the DNA of unidentified Rickettsia spp. DNA sequencing followed by phylogenetic analyses revealed that gltA sequences of Rickettsia spp. determined herein clustered with those of R. aeschlimannii  and Coxiella  for the first time from Pakistan. Endosymbionts are non-pathogenic mutualistic and/or commensal microbes, and they are also abundant in ticks. Main tick endosymbionts belong to the genera of Rickettsia, Francisella and Coxiella [Ehrlichia spp. were most commonly detected in ticks across all AEZs. Ticks co-infected with multiple pathogens might pose a greater risk to animals as well as humans because of increased risk of co-infections which would ultimately increase the clinical complexity of diseases. High co-infection rates of microorganisms in ticks highlights the need of such studies to be conducted on larger populations to further assess pathogen communities and their potential interactions as well as their pathogenic or zoonotic potential.In this study, a high prevalence of endosymbionts such as FLEs (91.5%) was inferred, for the first time, in m et al.  investigCoxiella , 77. EndCoxiella ; (ii) caCoxiella , 81; andCoxiella –84. The Coxiella , season Coxiella , geograpCoxiella , tick spCoxiella , tick liCoxiella , feedingCoxiella  and co-eCoxiella . Given tThis study reports that multiple TBPs of animal and public health significance are carried by bovine tick population in Pakistan. Co-infections with multiple pathogens are common, and endosymbionts are ubiquitously present. Overall, the prevalence of TBPs does not vary significantly across different AEZs of the country, but some pathogens may be restricted to particular regions or AEZs. Future studies are required to characterize endosymbionts further to explore their possible interaction(s) with pathogens transmitted by ticks collected from a larger animal population across different seasons in various AEZs.Additional file 1: Table S1. Primers used for validation of microfluidic real-time PCR results.Additional file 2: Figure S1. Multiple correspondence analyses maps from the projections of the first two dimensions, showing the associations between infections (co-occurrence), and their distributions among six districts in Punjab and Sindh provinces of Pakistan. Percentage in each dimension (axis) indicates the fraction of the inertia that each principal component explains. Analyses are shown for correlation between infections and their prevalence in different provinces.Additional file 3: Figure S2. Multiple correspondence analyses maps from the projections of the first two dimensions, showing the associations between infections (co-occurrence), and their distributions among six districts in Punjab and Sindh provinces of Pakistan. Percentage in each dimension (axis) indicates the fraction of the inertia that each principal component explains. Analyses are shown for correlation between infections and their prevalence in different districts.Additional file 4: Figure S3. Multiple correspondence analyses maps from the projections of the first two dimensions, showing the associations between infections (co-occurrence), and their distributions among six districts in Punjab and Sindh provinces of Pakistan. Percentage in each dimension (axis) indicates the fraction of the inertia that each principal component explains. Analyses are shown for correlation between infections and their prevalence in bovine host species.


Small fruits are a multi-billion dollar industry in the US, and are economically important in many other countries. However, they are perishable and susceptible to physiological disorders and biological damage. Food safety and fruit quality are the major concerns of the food chain from farm to consumer, especially with increasing regulations in recent years. At present, the industry depends on pesticides and fungicides to control food spoilage organisms. However, due to consumer concerns and increasing demand for safer produce, efforts are being made to identify eco-friendly compounds that can extend the shelf life of small fruits. Most volatiles and essential oils produced by plants are safe for humans and the environment, and lots of research has been conducted to test the in vitro efficacy of single-compound volatiles or multi-compound essential oils on various microorganisms. However, there are not many reports on their in vivo (in storage) and In situ (in the field) applications. In this review, we discuss the efficacy, minimum inhibitory concentrations, and mechanisms of action of volatiles and essential oils that control microorganisms (bacteria and fungi) on small fruits such as strawberries, raspberries, blueberries, blackberries, and grapes under the three conditions. There are two major concerns for the handling and consumption of small fruits. First is their short shelf life, and second is food safety. Fresh produce in particular is gaining increasing attention in the current food safety and regulatory climate. Centers for Disease Control and Prevention , has devStandard small fruit production practices discourage growers from washing fruits after harvest and during storage. This may contribute to the buildup of food-borne bacteria and may pose a food safety risk to consumers since small fruits are eaten fresh. Also, small fruits have a very short shelf life due to their susceptibility to spoilage pathogens, physiological disorders and mechanical injury . This is2 in modified atmospheres has been recommended to reduce spoilage [2 levels in storage, it causes off-flavors in the long term [Low temperature storage is not adequate to extend fruit shelf-life for prolonged storage or for delivery to distant markets. Therefore, controlled temperature combined with high COspoilage . Althougong term , and appong term ,7,8. Howong term ,8,9. Higong term  and raisong term . Other cong term . Therefoong term .Nowadays, consumers concerned about fungicide residues in small fruits demand safer alternatives to replace synthetic pesticides . TherefoNatural plant products tend to have low mammalian toxicity, broad-spectrum antimicrobial activity, are less hazardous than synthetic compounds, and are generally more acceptable to the public ,14,15,16Salmonella spp., Escherichia coli, and Listeria) and fruit pathogens (mainly Botrytis and Colletotrichum) on extending the shelf life of small fruits, such as strawberries (Fragaria × ananassa Duch), raspberries (Rubus spp.), blueberries (Vaccinium spp.) and grapes (Vitis vinifera L.). We also provide insight into their in vitro, in vivo (in storage) and In situ (in the field) applications, and their mechanisms of action. A summary of the papers reviewed is presented in In this review, we discuss the efficacy of volatiles (single compounds) and essential oils  produced by plants to control food-borne bacteria  to control foodborne pathogens, such as i dishes . Howeveri dishes  studied Citrus × bergamia Risso & Poit.) on table grapes (V. vinifera L.) during storage. They found that incorporation of bergamot essential oil improved the antimicrobial activity of the coatings and significantly reduced mold, yeast, and mesophile counts.Sánchez-González et al.  tested bPlant volatiles are mainly aldehydes, alcohols, acids and esters synthesized through the hydroperoxide lyase (HPL) and liposygenase (LOX) pathways. These products are involved in plant wounding responses and may have physiological roles  beside their role in aroma biosynthesis .Solanum sp. ‘Mountain Pride’) leaves were tested on conidiospores from Alternaria alternata and Botrytis cinerea. (E)-hex-2-enal and hexanal inhibited A. alternata hyphal growth and B. cinerea spore growth, while the terpene limonene and 2-carene did not affect hyphal growth [The effect of single compound terpene, terpenoids, and aldehyde vapors and their mixture from crushed tomato -hex-2-enal significantly inhibited mycelial growth and spore germination . Hexanal was only effective when applied in higher doses of up to 10 times [C. acutatum, B. cinerea, and A. alternata at low concentrations of 1.1, 1.3 and 2.3 μL L−1 air respectively. Andersen et al. [−1 air of hexanal reduced A. alternata growth on agar by 50%. Song et al. [−1 air inhibited B. cinerea growth on potato dextrose agar (PDA ) media and Archbold et al. [−1 (2 μL/250 ml container) hexanal reducing B. cinerea in inoculated strawberries by 90%. In contrast to all these reports, Hamilton-Kemp et al. [A. alternata and B. cinerea mycelial growth at low levels.Similarly, Arroyo et al.  examined10 times . The inh10 times  observedn et al.  reportedd et al.  showed 0p et al.  observedA. alternata, B. cinerea, and Colletotrichum gloeosporioides) species in vitro [In an experiment, among five antifungal volatiles -hex-3-en-1-ol and (E)-hex-2-enal) released by raspberry and strawberry fruits, (E)-hex-2-enal and benzaldehyde were the most effective against three fungal  and then evaluated in vitro against diseases .Essential oils (EO) are naturally occurring substances extracted from plant material and are a complex of volatiles produced in different plant parts. They constitute different plant secondary metabolites mainly from terpenes, and other aromatic compounds . They haEssential oils have three main features. Since they are produced by plants, they are safer to the environment and humans than synthetic alternatives. Second, they have low risk of resistance development by pathogens because they are a mixture of oil components and pathogens lack the mechanism to develop resistance against a range of chemicals. Third, there is a broad range of natural compounds that have the potential to be used for controlling pathogens . EssentiA large number of papers have been published on the biological activity of essential oils. The large variability among the data may be attributed to factors such as genetics, climate, geographical and seasonal conditions, distillation techniques and time of harvest . ConsequB. cinerea or other small fruit pathogens [Most of the reports on the inhibition of post-harvest fungal pathogens by essential oils focus on in vitro conditions . The effathogens ,6,7,18.B. cinerea. Wilson et al. [Capsicum and Allium genera were the most effective and inhibited B. cinerea spore germination after 48 and 24 hours, respectively. Among 49 essential oils tested, red thyme (Thymus zygis L.), palmarosa (Cymbopogon martini (Roxb.) Will. Watson), clove buds (Syzygium aromaticum (L.) Merr. & L.M. Perry), and cinnamon leaf (Cinnamomum zeylanicum Blume) greatly inhibited germination of B. cinerea spores at the lowest concentration tested . The essential oils with high antifungal activity and the highest frequency were: cineole, limonene, α-pinene, β-myrcene, camphor and β-pinene [Two studies ,18 screeZingiber officinale Roscoe, Z. cassumunar Roxb., Prunus persica (L.) Batsch, Ocimum sanctum L., Ocimum gratissimum L., Ocimum canum Sims, Lawsonia inermis L., Eupatorium cannabinum L., Eucalyptus citriodora Hook., and Chenopodium ambrosioides L.) showed 100% toxicity at 500 (mg L−1). The essential oils were a mixture of nine major and sixteen minor compounds with possible synergistic effects between major and minor constituents [Tripathi et al.  tested etituents .B. cinerea mycelium was completely inhibited by thyme, oregano, dictamnus and marjoram essential oils at 150, 200, 200 and 300 mgml-1, respectively. The main component of oregano oil was thymol, and the main component of marjoram, thyme and dictamnus oils was carvacrol [In another study, growth of arvacrol .Thymus vulgaris  against two strawberry storage pathogens (Rhizopus stolonifer and B. cinerea). Oil from Laval-2 clones showed higher antifungal activity in vitro and decay control action in vivo which is mainly due to its higher carvacrol, thymol and linalool contents. Lemongrass oil reduced germ tube length and spore germination in R. stolonifer, Colletotrichum coccodes, and B. cinerea [Bhaskara Reddy et al.  reported cinerea .B. cinerea isolated from grapes. B. cinerea was the most susceptible to cinnamon, thyme, and eugenol at the lowest concentration tested (500 μL L−1).Combrinck et al.  tested eC. acutatum (667 μL L−1 of medium) and their volatiles disabled appressoria formation (1.53 μL L−1 of air) and prevented conidia germination .In the study conducted by Duduk et al. , clove bOriganum vulgare L. essential oil on Aspergillus niger and R. stolonifer on grapes. Mycelial growth was inhibited by essential oil in in vitro conditions.Santos et al.  tested tB. cinerea. Thymus glandulosus Req. and Origanum compactum Benth., with thymol and carvacrol as main compounds, inhibited mycelium growth more effectively than others.Bouchra et al.  evaluateDifferent concentrations of hexanal are effective against fungal growth. Caccioni et al.  believedA. alternata and C. acutatum growth (3.0 and 3.3 μL respectively). Therefore, if media can absorb henxanal, we hypothesize that fresh produce can also absorb small amounts of volatiles and extend their shelf life by extruding it. Gardini et al.  posited B. cinerea and A. alternata hyphal growth than hexanal . Arroyo et al. [C. acutatum mycelial growth [C. acutatum to volatiles compared to B. cinerea and A. alternata as was mentioned by Almenar et al. [C. acutatum treated with (E)-hex-2-enal by transmission electron microscopy. They observed that cell components were highly disorganized and cell wall and plasma membrane were disrupted, resulting in lysis of organelles and cell death. The saturation status of volatile compounds seems to also affect their efficacy with unsaturated compounds being more reactive than saturated ones. Hamilton-Kemp et al.  and Andeo et al.  showed tl growth . The rear et al. . Arroyo r et al.  studied B. cinerea and A. alternata reacted to hexanal similarly but different from C. acutatum. They suggested that the inhibition mechanisms are different, but no further explanation was provided. However, Hamilton-Kemp et al. [C. acutatum and B. cinerea responded similarly meaning hyphal growth was inhibited by aldehyde but not terpenes. However, they suggested that more cautious interpretation is needed as variable culture conditions and organism strain may affect the results.Almenar et al.  reportedp et al.  reportedThe difference in the affinity of these compounds with microbial membranes seems to affect their effectiveness and their toxicity . Cell meB. cinerea exposed to volatiles of grape  resulting in cell wall deformation. Hyphae from the control treatment was healthy, possessing conidiophores with abundant conidia [In this regard, Kulakiotu et al.  reported conidia . The mec conidia .The fact that different natural compounds have a different antifungal profile suggests that different modes of action exist. In human pathogens, several mechanisms of actions have been reported for antifungal activity of natural plant compounds. Sivakumar et al.  reportedAldehydes, another group of volatiles, inhibit fungal cell division by reacting with, and inactivating sulfhydryl, the functional group involved in fungal cell division. Some aldehydes  are good electron acceptors and form a charge transfer complex with electron donors present in fungal cells, thus interfering with fungal metabolism .C. acutatum, Colletotrichum fragariae, C. gloeosporioides, Fusarium oxysporum, B. cinerea, and Phomopsis sp. Nevertheless, the accessibility of α,β-unsaturated carbonyl groups to bulky nucleophile biomolecules plays an important role in their antifungal activity [Volatiles with α,β-unsaturated cabonyl groups  often react with nucleophilus in fungi through the Michael reaction to create chemical modifications and interrupt fungal growth  of C. acactivity .C. ambrosioides essential oil component such as carvacrol [2+ and reduced hemin [2+ permeable transient receptor potential channels, inhibits sarcoplasmic reticulum Ca2+ ATPase, and activates ryanodine receptors in skeletal muscle, thereby influencing intracellular calcium homeostasis -hex-2-enal in inoculated strawberry fruit. This compound also effectively reduced B. cinerea disease symptoms in grapes, blackberry (Rubus spp.) and strawberry [A. alternata, B. cinerea, and C. gloeosporioides on strawberry and raspberry fruits.Arroyo et al.  reportedrawberry . Vaughn rawberry , evaluatB. cinerea activity and inoculum density, thereby limiting incidence and infection in kiwifruits [In another study, treatments with thymol, eugenol, and menthol reduced decay in strawberries, with thymol being the most effective at slowing berry decay compared to the other two compounds . Similarwifruits .Eucalyptus globulus L.) and cinnamon oil  improved fruit quality and reduced fruit decay [C. acutatum and B. cinerea penetration, development and number of infected fruits at concentrations above 76.5 μL L−1 of air [T. vulgaris L.) EO had higher antifungal activity and suppressed pathogen development at concentrations above 15.3 μL L−1 of air. At a higher concentration (153 μL L−1 of air), thyme EO inhibited C. acutatum development on inoculated fruits [R. stolonifer and B. cinerea was controlled by volatiles of T. vulgaris [Essential oils from a number of plants have shown activity against strawberry pathogens. For example, eucalyptus  Merr. & L.M. Perry), thyme, and massoialactone (derived from the bark of the tree Cryptocarya massoia R.Br.) significantly reduced necrotic lesions on grape leaves caused by B. cinerea when treated with thyme, and massoialactone. In a separate study, Valero et al. [Essential oils have also been tested on grape diseases. In a laboratory study, Walter et al.  found tho et al.  showed tO. vulgare essential oil changed spore and mycelia morphology and inhibited growth of A. niger, and R. stolonifer spore germination on inoculated grapes in storage [Z. officinale, O. sanctum and P. persica were also effective in controlling storage rot of grapes during in vivo trials, with treated grapes showing an improved shelf life for up to 6 days [Chitosan packages infused with  storage . The oilo 6 days .Wang et al.  found thCarvacrol, anethole, and perillaldehyde also enhanced antioxidant activity and increased total anthocyanins, phenolics and hydroxyl radical (•OH) scavenging capacity in blueberry fruit tissues . ChitosaSimilarly, postharvest essential oil treatments  enhanced antioxidant capacity in raspberries with perillaldehyde being the most effective . Gerbera jamesonii cv. Dune) flowers treated with thymol [Grape berries in a modified atmosphere package with eugenol or thymol had lower weight loss, lower changes in skin color, reduced ripening, and lower decay throughout storage . Lower wh thymol .O. vulgare essential oil also preserved the quality, physical, physiochemical and sensory attributes of grapes in storage [Sánchez-González et al.  tested c storage .Plant volatiles play a major role in self-defense. Pe´rez et al.  observedArabidopsis thaliana, (E)-hex-2-enal activated several self-defense genes , lignified leaves and accumulated pathogenesis-related proteins , which reveals that volatile treatments stimulate a wound-repair mechanism, and will ultimately act as a physical barrier to the pathogen penetration [Actinidia deliciosa cv. ‘Hayward’) against B. cinerea with the application of grape (cv. Isabella) volatiles which induced mechanisms of resistance in the host [Six and nine-carbon volatiles are produced in plant tissues in response to wounding in LOX (liposygenase) and HPL (hydroperoxide lyase) pathways. In addition to their role in aroma biosynthesis, products of LOX and HPL pathways, have antimicrobial and antifungal activities and may have a role in plant-pathogen interactions. Some have also shown a stimulatory effect on some pathogenic fungi. In etration . Externaetration . This hathe host .Since plant volatiles inhibit fungal growth and are produced rapidly through the lipoxygenase pathway (in response to wounding), more studies are needed to further clarify their role in plant self-defense systems.B. cinerea hyphae treated with grape (cv. Isabella) volatiles [In addition to self-defense and induced resistance, essential oils affect pathogens by contact either in media or as volatiles in storage. Essential oils also lead to the secretion of cell protoplasts without previous cell wall disruption, as shown by the presence of chlamydospores and deformation of cell walls in olatiles . The bioactivity of essential oils in the vapor phase is a very attractive characteristic that makes them suitable for use as fumigants in small fruits not suited to aqueous sanitation . Easy apExtended exposure to volatiles has resulted in phytotoxicity on strawberry fruits and affected fruit quality . The phyT. vulgaris essential oils. Further work is needed to identify and minimize the cause of adverse effects on fruit quality by volatiles [In the authors’ preliminary experiments with the application of thymol and carvacrol in air-tight containers, phytotoxicity was observed as increased mushiness and dullness of strawberry fruits after 24 hours (unpublished data). However, Bhaskara Reddy et al.  did not olatiles .Very few studies have reported on the sensory characteristics of fruits treated with essential oils. Neri et al.  reportedThe reviewed articles suggest that most of the essential oils do not leave off-flavors or off-odors on intact fruits at the minimum inhibitory concentrations, especially when stored fruits finish their ripening process. However, there is variation between fruit species and cultivars and each commodity has to be tested individually. For example, Neri et al. , reporteB. cinerea in the field trial [To our knowledge, only one research paper described the application of essential oils in the field. A single application of thyme oil or massoialactone application on grape bunches and leaves reduced ld trial . Howeverld trial .In this review, the efficacy of essential oils (plant-based multi-compounds) and single compound volatiles was discussed in in vitro, in vivo (in storage) and In situ (in the field) conditions. Despite the importance of fresh fruits food safety, there are not many research articles on the effectiveness of essential oils to control bacteria populations on small fruits, which suggests the need for research in this area. Many research articles discussed the positive effects of volatiles and essential oils in in vitro conditions on media-grown fungal populations with a large variation in their efficacy and their minimum inhibitory concentrations. Most of these variations were related to the vapor pressure of volatiles. The efficacy of multi-compound essential oils is even more variable due to genetic variation, environmental conditions, and synergistic effects of multiple compounds. Major compounds play an essential role in the antimicrobial activity of essential oils, however, minor compounds in the mixture may have synergistic effects. Future studies are needed to explain the role of single natural compounds or their combination in plant-fungal interactions.(E)-hex-2-enal and hexanal were the most effective volatiles and thyme the most effective essential oil in controlling fungal diseases of small fruits both in vitro and in storage. Thyme oil has the potential as a postharvest treatment to extend shelf life of fresh fruits and vegetable and to replace commercial fungicides or controlled atmosphere storage. However, active packaging and improved formulation techniques are needed to prolong activity, reduce volatility and improve coverage.Several researchers have mentioned the toxicity of volatiles and essential oils after extended exposure. Research is needed to study the effect of volatiles and essential oils on vegetative and reproductive phases of fungal development, fruit quality , cell wall structure and integrity and their role in plant self-defense and postharvest storage.