Assessment of Zoonotic Transmission of Giardia and Cryptosporidium between Cattle and Humans in Rural Villages in Bangladesh

Giardia and Cryptosporidium are important causes of diarrhoea in Bangladesh. The high prevalence of both parasites in humans and cattle in rural Bangladesh and the common use of water ponds by village inhabitants and their animals suggest a potential for zoonotic transmission. Direct transmission of Giardia and Cryptosporidium between cattle and their handlers and indirect transmission through water ponds was investigated. Faecal/stool samples were collected from 623 calves and 125 calf handlers in a cross-sectional survey. In two villages, water samples were collected monthly from water ponds and faecal/stool samples were collected monthly from inhabitants and their cattle. Giardia cysts and Cryptosporidium oocysts were detected in water samples and in faecal/stool samples and positive samples were genotyped, to determine their human or animal origin. The prevalence of Giardia and Cryptosporidium in calves was 22% and 5% respectively. In calf handlers, the prevalence of Giardia and Cryptosporidium was 11.2% and 3.2% respectively. Both in the cross-sectional survey and in the longitudinal study in the villages, G. duodenalis assemblage E was most prevalent in calves, while in humans assemblage AII, BIII and BIV were found. In cattle, Cryptosporidium parvum, C. bovis and C. andersoni were identified, but no Cryptosporidium sequences were obtained from humans. Giardia and Cryptosporidium were detected in 14/24 and 12/24 water samples respectively. G. duodenalis assemblage E and BIV (-like), as well as C. andersoni and C. hominis were identified. Although the presence of Giardia and Cryptosporidium in both water ponds suggests that water-borne transmission of Giardia and Cryptosporidium is possible, the genotyping results indicate that there is no significant direct or indirect (water-borne) transmission of Giardia between cattle and people in this area of rural Bangladesh. No conclusions could be drawn for Cryptosporidium, because of the low number of sequences that were obtained from human and water samples.     

Fishborne Trematode Metacercariae in Freshwater Fish from Guangxi Zhuang Autonomous Region, China

A survey was performed to investigate the infection status of fishborne trematode (FBT) metacercariae in freshwater fish from Guangxi Zhuang Autonomous Region, China. A total of 307 freshwater fish of 31 species were collected from 5 administrative regions of Guangxi Zhuang Autonomous Region. They were examined by artificial digestion method from July 2003 to August 2004. No metacercariae were detected in fish from Fusui-xian. In fish from Mashan-xian and a market in Nanning, 3 species of metacercariae, Haplorchis taichui, Haplorchis pumilio, and Centrocestus formosanus, were mainly detected. Metacercariae (8 in number) of Clonorchis sinensis were found in 1 Chanodichthys dabryi purchased from a market in Nanning. In fish from Yangshuo, Metagonimus yokogawai metacercariae were detected from all 18 fish species examined. Total 13 C. sinensis metacercariae were found in 3 out of 10 Hemibarbus maculatus from Yangshuo. All 7 Zacco platypus from Yangshuo were infected with 8-112 Echinochasmus perfoliatus metacercariae. In fish from Binyang-xian, H. pumilo metacercariae were mainly detected in all 5 fish species examined, and only 1 metacercaria of C. sinensis was found in a Hemiculter leucisculus. From the above results, it was confirmed that some species of freshwater fish play a role of second intermediate hosts for FBT in Guangxi Zhuang Autonomous Region, China. In particular, 4 species of intestinal flukes, M. yokogawai, H. taichui, H. pumilio, and C. formosanus, were prevalent in fish hosts, whereas C. sinensis metacercariae were detected only in 3 fish species.     

Genetic Diversity of Cryptosporidium spp. in Captive Reptiles

The genetic diversity of Cryptosporidium in reptiles was analyzed by PCR-restriction fragment length polymorphism and sequence analysis of the small subunit rRNA gene. A total of 123 samples were analyzed, of which 48 snake samples, 24 lizard samples, and 3 tortoise samples were positive for Cryptosporidium. Nine different types of Cryptosporidium were found, including Cryptosporidium serpentis, Cryptosporidium desert monitor genotype, Cryptosporidium muris, Cryptosporidium parvum bovine and mouse genotypes, one C. serpentis-like parasite in a lizard, two new Cryptosporidium spp. in snakes, and one new Cryptosporidium sp. in tortoises. C. serpentis and the desert monitor genotype were the most common parasites and were found in both snakes and lizards, whereas the C. muris and C. parvum parasites detected were probably the result of ingestion of infected rodents. Sequence and biologic characterizations indicated that the desert monitor genotype was Cryptosporidium saurophilum. Two host-adapted C. serpentis genotypes were found in snakes and lizards.     

Leishmania (Sauroleishmania) tarentolae isolation and sympatric occurrence with Leishmania (Leishmania) infantum in geckoes,dogs and sand flies

The trypanosomatid protist Leishmania tarentolae is a saurian-associated parasite vectored by the Sergentomyia minuta sand fly. This study aimed to confirm the circulation of L. infantum and L. tarentolae in sand flies, reptiles and dogs and to isolate new strains of these protists. Reptilian and sheltered dog blood samples were collected, and sand flies were captured. Samples were tested for Leishmania spp. using duplex real-time PCR (dqPCR) and real-time PCR (qPCR); the origin of blood meal was identified in engorged sand flies by conventional PCR. The reptilian blood and intestinal content of sand fly females were cultured. Dog sera were tested by IFAT using both Leishmania species. Four Tarentola mauritanica geckoes were molecularly positive for L. infantum or L. tarentolae, with no co-infections; moreover, amastigote-like forms of L. infantum were observed in the bone marrow. 24/294 sand flies scored positive for Leishmania spp. by dqPCR, 21 S. minuta and two Phlebotomus perniciosus were positive for L. tarentolae, while only a single Ph. perniciosus was positive for L. infantum. Blood meal analysis confirmed reptile and dog in S. minuta, dog and human in Ph. perniciosus and dog in Phlebotomus neglectus. Two axenic strains of L. tarentolae were obtained. Twelve of 19 dogs scored positive for L. infantum and L. tarentolae by IFAT and three of them also for L. infantum by dqPCR, and six by qPCR. These data confirm the sympatric circulation of L. infantum and L. tarentolae in geckoes, sand flies, and dogs, and suggest that geckoes may be infected with L. infantum.     

Symbiotic microbial studies in diverse populations of Aphis gossypii,existing on altered host plants in different localities during different times

Complex interactions between symbiotic bacteria and insects ultimately result in equilibrium in all aspects of life in natural insect populations. In this study, abundance of principal symbiotic bacteria was estimated using qPCR in 1553 individuals of aphids, Aphis gossypii. Aphids were sampled from primary and secondary host plants—hibiscus and cotton. Hibiscus aphids were collected from 24 different locations in April, September, and November, whereas cotton aphids were collected between 2015 and 2017 from areas with wide variations in climatic conditions. About 30%–45% aphids were recorded with the most dominant symbiont, Arsenophonus. The other symbionts were in low frequency, and about 7% of aphids were noted with Hamiltonella, Acinetobacter, and Microbacterium, and 3% of aphids were verified with Serratia and Pseudomonas. Aphids infected with Hamiltonella, Arsenophonus, and Serratia can influence Buchnera densities. Hamiltonella has positive interaction with densities of Arsenophonus and Serratia. Almost 100% coinfection of Hamiltonella and Arsenophonus was detected in Xinxiang aphids and 50% coinfection was reported in aphids from North China, while no coinfection was detected in Hainan aphids. These findings describe the prevalence pattern and richness of core community of symbiotic bacteria in naturally occurring populations of A. gossypii and provide new insights for the study of symbiotic bacteria.     

Identification and characterization of four new tra cistrons on the E. coli K12 sex factor F.

Thirty-eight amber-suppressible transfer-deficient mutants of the Flac element F13-1 have been analyzed in conjugational complementation tests. Many of the mutations were in tra genes that have been identified previously (including traI), but several were in four new genes, traN, traU, traV, and traW. Results for representative mutations in the new genes were confirmed by complementation tests using λtra transducing phages and these also allowed the genes to be mapped quickly and simply. Approximate physical locations were found by complementation with a series of chimeric plasmids carrying various EcoRI fragments of the F transfer region. All four genes are located in the transfer operon, the order being traA … traBtraVtraWtraCtraUtraNtraF … traD. While traU, traV, and traW mutants were resistant to male-specific phages, traN mutants were sensitive.     

Distribution and diversity of rhizobia associated with wild soybean (Glycine soja Sieb. & Zucc.) in Northwest China

A total of 155 nodule isolates that originated from seven sites in Northwest China were characterized by PCR-RFLP of the 16S rRNA gene and sequence analysis of multiple core genes (16S rRNA, recA, atpD, and glnII) in order to investigate the diversity and biogeography of Glycine soja-nodulating rhizobia. Among the isolates, 80 were Ensifer fredii, 19 were Ensifer morelense, 49 were Rhizobium radiobacter, and 7 were putative novel Rhizobium species. The phylogenies of E. fredii and E. morelense isolates in a concatenate tree (assembly of all housekeeping genes) were generally consistent with those in individual gene trees. However, incongruence was found in the phylogenies of the different genes of Rhizobium isolates, indicating that lateral transfer or recombination possibly occurred in these gene loci. Despite their species identity, all the isolates in this study formed a single lineage related to E. fredii in nodAand nifH gene phylogenies, which also indicated that the symbiotic genes were laterally transferred between different species. Biogeographic patterns were found at the species and strain genomic type levels, as revealed by BOXA1R fingerprinting, demonstrating that the evolution of rhizobial populations in different geographic locations was related to soil types, altitude and spatial effects. This study is the first to report that E. morelense, R. radiobacter, and Rhizobium sp. are microsymbionts of G. soja, as well as showing that the diversity of G. soja rhizobia is enhanced and new rhizobia have evolved in Northwest China.     

Fishborne Trematode Metacercariae Detected in Freshwater Fish from Vientiane Municipality and Savannakhet Province, Lao PDR

Freshwater fish from Vientiane Municipality and Savannakhet Province, Lao PDR were examined by the muscle compression and artificial digestion methods to know the infection status with trematode metacercariae. In the fish from Savannakhet, 2 species of metacercariae, Opisthorchis viverrini and Haplorchis taichui, were detected. O. viverrini metacercariae were found in 6 species of fish, Puntius brevis, Hampala dispar, Esomus metallicus, Mystacoleucus marginatus, Puntioplites falcifer, and Cyclocheilichthys armatus. H. taichui metacercariae were detected in 3 species of fish, P. brevis, P. falcifer, and M. marginatus. In the fish from Vientiane, 4 species of metacercariae, O. viverrini, H. taichui, Haplorchis yokogawai, and Centrocestus formosanus, were detected. Among them, O. viverrini metacercariae were found in 7 species of fish, Onychostoma elongatum, C. armatus, H. dispar, P. brevis, Cyclocheilichthys repasson, Osteochilus hasseltii, and Hypsibarbus lagleri. The metacercariae of H. taichui were detected in 6 species of fish, C. repasson, O. elongatum, C. armatus, H. dispar, Labiobarbus leptocheila, and Cirrhinus molitorella. The metacercariae of H. yokogawai were found in 9 species of fish, C. repasson, O. elongatum, C. armatus, H. dispar, Labiobarbus leptocheila, O. hasseltii. C. molitorella, Hypsibarbus wetmorei, and H. lagleri. The metacercariae of C. formosanus were detected in 4 species of fish, C. repasson, P. brevis, O. hasseltii, and C. molitorella. From these results, it is confirmed that fishborne trematode metacercariae, i.e. O. viverrini, H. taichui, H. yokogawai and C. formosanus, are prevalent in various species of freshwater fish from Savannakhet Province and Vientiane Municipality, Lao PDR.     

Mycoflora isolation and molecular characterization of Aspergillus and Fusarium species in Tunisian cereals

Wheat, barley and maize are the mainly consumed cereals in Tunisia. This study aimed to determine the mycoflora of these cereals with special focus on the mycotoxigenic Aspergillus and Fusarium species. Freshly harvested samples and other stored samples of each type of cereal (31 and 34 samples, respectively) were collected in Tunisia and cultured for fungal isolation and identification. Identification of fungal genera was based on morphological features. Aspergillus and Fusarium species were identified by species specific PCR assays complemented with DNA sequencing. Alternaria (70.83%), Eurotium (62.50%), Aspergillus (54.17%) and Penicillium (41.67%) were the most frequent fungi isolated from wheat. Penicillium (75%), Aspergillus (70%), Eurotium (65%) and Alternaria (65%) were the most frequently recovered genera from barley. The predominant genera in maize were Aspergillus (76.19%), Eurotium (42.86%), and Penicillium (38.09%). Aspergilllus, Penicillium, Fusarium and Alternaria were detected in both stored and freshly harvested grain samples. The frequencies of contamination with Aspergillus, Fusarium and Alternaria were higher in freshly harvested samples, whereas Penicillium species were more frequent in stored samples. The predominant Aspergillus species detected were A. flavus and A. niger. The Fusarium species detected were F. equiseti, F. verticillioides, F. nygamai, and F. oxysporum. This study suggested the potential risk for Aflatoxins and, to a lesser extent, for Ochratoxin A in Tunisian cereals. This is the first survey about mycoflora associated with wheat, barley and maize in Tunisia.     

The roots of the halophyte Salicornia brachiata are a source of new halotolerant diazotrophic bacteria with plant growth-promoting potential

Soil salinity is the major cause limiting plant productivity worldwide. Nitrogen-fixing bacteria were enriched and characterised from roots of Salicornia brachiata, an extreme halophyte which has substantial economic value as a bioresource of diverse and valuable products. Nitrogen-free semisolid NFb medium with malate as carbon source and up to 4% NaCl were used for enrichment and isolation of diazotrophic bacteria. The isolates were tested for plant growth-promoting traits and 16S rRNA, nifH and acdS genes were analysed. For selected strains, plant growth-promoting activities were tested in axenically grown Salicornia seedlings at different NaCl concentrations (0–0.5M). New halotolerant diazotrophic bacteria were isolated from roots of S. brachiata. The isolates were identified as Brachybacterium saurashtrense sp. nov., Zhihengliuella sp., Brevibacterium casei, Haererehalobacter sp., Halomonas sp., Vibrio sp., Cronobacter sakazakii, Pseudomonas spp., Rhizobium radiobacter, and Mesorhizobium sp. Nitrogen fixation as well as plant growth-promoting traits such as indole acetic acid (IAA) production, phosphate solubilisation, and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity were demonstrated. For Brachybacterium saurashtrense and Pseudomonas sp., significant plant growth-promoting activities were observed in Salicornia in salt stress conditions. Salicornia brachiata is a useful source of new halotolerant diazotrophic bacteria with plant growth-promoting potential.     

Expression Stabilities of Candidate Reference Genes for RT-qPCR under Different Stress Conditions in Soybean

Due to its accuracy, sensitivity and high throughput, real time quantitative PCR (RT-qPCR) has been widely used in analysing gene expression. The quality of data from such analyses is affected by the quality of reference genes used. Expression stabilities for nine candidate reference genes widely used in soybean were evaluated under different stresses in this study. Our results showed that EF1A and ACT11 were the best under salinity stress, TUB4, TUA5 and EF1A were the best under drought stress, ACT11 and UKN2 were the best under dark treatment, and EF1B and UKN2 were the best under virus infection. EF1B and UKN2 were the top two genes which can be reliably used in all of the stress conditions assessed.     

Antagonists of Plant-parasitic Nematodes in Florida Citrus

In a survey of antagonists of nematodes in 27 citrus groves, each with a history of Tylenchulus semipenetrans infestation, and 17 noncitrus habitats in Florida, approximately 24 species of microbial antagonists capable of attacking vermiform stages of Radopholus citrophilus were recovered. Eleven of these microbes and a species of Pasteuria also were observed attacking vermiform stages of T. semipenetrans. Verticillium chlamydosporium, Paecilomyces lilacinus, P. marquandii, Streptomyces sp., Arthrobotrys oligospora, and Dactylella ellipsospora were found infecting T. semipenetrans egg masses. Two species of nematophagous amoebae, five species of predatory nematodes, and 29 species of nematophagous arthropods also were detected. Nematode-trapping fungi and nematophagous arthropods were common inhabitants of citrus groves with a history of citrus nematode infestation; however, obligate parasites of nematodes were rare.     

Fungal diversity in ancient documents. A case study on the Archive of the University of Coimbra

This multidisciplinary research combines knowledge in molecular biology with fungal morphology, aiming at the identification of infecting fungi from historical documents on the Archive of the University of Coimbra. The identification of infecting fungi on several bibliographic documents and support materials was based both on ribosomal DNA loci amplification and sequencing, and morphological identification, using macro- and microscopical traits. A high fungal diversity was found in all types of support: parchment, laid-paper and wood-pulp paper. Fourteen fungal genera were isolated, identified, and kept in culture. The most frequent were Cladosporium, Penicillium and Aspergillus, and other less frequent genera, such as Alternaria, Botrytis, Chaetomium, Chromelosporium, Epicoccum, Phlebiopsys and Toxicocladosporium were also present. Within these genera, 20 different species were identified, from which 15 were found only in a single support type. Cladosporium cladosporioides and Penicillium chrysogenum were the only species present in all support types.     

Alkylpyrazines in ponerine ants: Their presence in three genera,and caste specific behavioural responses to them in Odontomachus troglodytes

Alkylpyrazines were found in the mandibular gland secretions of three Ethiopian ponerine ants. The major components in Odontomachus troglodytes were 2,6-dimethyl-3-n-butyl- and n-pentylpyrazine; in Anochetus sedilloti 2,6-dimethyl-3-sec-butyl- and 2,5-dimethyl-3-n-pentylpyrazine, and in Brachyponera sennaarensis 2,6-dimethyl-3-n-butyl and n-pentylpyrazines. The male caste in O. troglodytes contained 2,6-dimethyl-3-n-hexyl- and n-butylpyrazine. In O. troglodytes caste-specific behavioural differences were found between males and workers in response to gland equivalent quantities of their pheromones. The fragile males retreated from the alkylpyrazines whereas the workers were attracted to and attacked the pheromone source. Large quantities (5 gland equivalents) of pheromones were also repellent to the workers.     

Association of Borrelia garinii and B. valaisiana with Songbirds in Slovakia

In Europe, 6 of the 11 genospecies of Borrelia burgdorferi sensu lato are prevalent in questing Ixodes ricinus ticks. In most parts of Central Europe, B. afzelii, B. garinii, and B. valaisiana are the most frequent species, whereas B. burgdorferi sensu stricto, B. bissettii, and B. lusitaniae are rare. Previously, it has been shown that B. afzelii is associated with European rodents. Therefore, the aim of this study was to identify reservoir hosts of B. garinii and B. valaisiana in Slovakia. Songbirds were captured in a woodland near Bratislava and investigated for engorged ticks. Questing I. ricinus ticks were collected in the same region. Both tick pools were analyzed for spirochete infections by PCR, followed by DNA-DNA hybridization and, for a subsample, by nucleotide sequencing. Three of the 17 captured songbird species were infested with spirochete-infected ticks. Spirochetes in ticks that had fed on birds were genotyped as B. garinii and B. valaisiana, whereas questing ticks were infected with B. afzelii, B. garinii, and B. valaisiana. Furthermore, identical ospA alleles of B. garinii were found in ticks that had fed on the birds and in questing ticks. The data show that songbirds are reservoir hosts of B. garinii and B. valaisiana but not of B. afzelii. This and previous studies confirm that B. burgdorferi sensu lato is host associated and that this bacterial species complex contains different ecotypes.     

The interaction of Babesia caballi kinetes with tick cells

Babesia caballi caused pathological changes in the vector tick, Anocentor nitents. These included the loss of hemocytes, reddish-brown discoloration of the hemolymph, retardation in clotting, and reduction in egg production. Primary cultures were initiated from A. nitens embryos transovarially infected with B. caballi. Cell lines that were isolated were morphologically similar and composed of stellate, fusiform, and hemocyte-like cells, but none was visibly infected with B. caballi. Primary cultures from heavily infected ticks were not viable and did not develop into lines. Kinetes from hemolymph and organs of A. nitens were cocultivated with cell lines isolated from embryos of A. nitens, Rhipicephalus appendiculatus, and Rhipicephalus sanguineus. They attached to and actively penetrated the cells. Cells that were penetrated by or infected with B. caballi showed rounding off, degeneration, and lysis. In Giemsa-stained spreads prepared after 1 or 2 days in vitro, more kinetes were associated with R. appendiculatus cells when the medium contained A. nitens egg extract (TEE). Ninety percent of the B. caballi kinetes were always found within or in close contact with cells from A. nitens, cultured with or without TEE. The parasites were club shaped, amoeboid, or round. Intracellular Babesia were frequently lying adjacent to the host cell nucleus. In the cultures the parasites were detected by light microscopy for 3 to 5 days; they did not transform into other tick-associated stages and were not seen after 1 week. This is the first report of the cultivation of tick stages of B. caballi in tick cell cultures.     

Plant-Parasitic Nematodes of South Viet Nam

Between 1974 and 1978, 2,842 identifications of plant-parasitic nematodes were made from more than 1,700 soil and plant samples collected in eight provinces of South Viet Nam. Species in nine genera—Helicotylenchus, Criconemoides, Meloidogyne, Pratylenchus, Tylenchorhynchus, Hoplolaimus, Hirschmanniella, Xiphinema, and Rotylenchulus—comprised 96.1% of the identifications; the remaining 3.9% were species of 11 genera. Fourteen genera were associated with rice which was grown on about 2,500,000 ha in 1970. Of these, Ditylenchus, Hirschmanniella, and Meloidogyne were most important. Ditylenchus angustus caused severe damage to about 50,000 ha of flooded rice in the Mekong Delta in 1976. Hirschmanniella spp. were found in all samples examined from flooded rice fields. Meloidogyne spp. were common in rice seedbeds, upland rice, and rice not kept flooded continuously. Meloidogyne and Pratylenchus spp. were found in roots of 22 of the 32 crop plants sampled. Little or no attempt was made in South Viet Nam to control nematodes.     

Soil pH Effects on Nematode Populations Associated with Soybeans

''Amsoy'' soybeans were grown for 2 months in nonsterilized Jackson silt loam amended to pH 4.0, 6.0, and 8.0. Nematodes were extracted biweekly from soil and roots. The greatest numbers of Pratylenchus alleni colonized soybean roots at pH 6.0. Hoplolaimus galeatus and members of the Tylenchinae-Psilenchinae survived best at pH 6.0, while numbers o f the Dorylaimoidea were greatest at both pH 6.0 and 8.0. The non-stylet nematodes were recovered in greater numbers from pH 8.0 soil. Potassium, manganese, and phenols were highest in soybean plants grown in pH 4.0 soil, the pH at which there were the fewest nematodes. A thicker suberized outer layer o f root tissue occurred in plants grown at pH 4.0.     

Comparative analysis of bacterial community and antibiotic-resistant strains in different developmental stages of the housefly (Musca domestica)

The housefly (Musca domestica) is an important host for a variety of bacteria, including some pathogenic and antibiotic-resistant strains. To further investigate the relationship between the housefly and the bacteria it harbors, it is necessary to understand the fate of microorganisms during the larval metamorphosis. The major bacterial communities in three developmental stages of the housefly (maggot, pupa, and adult fly) were investigated by a culture-independent method, polymerase chain reaction–denaturing gradient gel electrophoresis (PCR?DGGE) analysis of 16S rRNA genes. The bacteria that were identified using DGGE analysis spanned phyla Proteobacteria, Firmicutes, and Bacteroidetes. Changes in the predominant genera were observed during the housefly development. Bacteroides, Koukoulia, and Schineria were detected in maggots, Neisseria in pupae, and Macrococcus, Lactococcus, and Kurthia in adult flies. Antibiotic-resistant bacteria were screened using a selective medium and tested for antibiotic susceptibility. Most resistant isolates from maggots and pupae were classified as Proteus spp., while those from adult flies were much more diverse and spanned 12 genera. Among 20 tested strains across the three stages, 18 were resistant to at least two antibiotics. Overall, we demonstrated that there are changes in the major bacterial communities and antibiotic-resistant strains as the housefly develops.