Retinal ganglion cells (RGCs) convey the major output of information collected from the eye to the brain. Thirty subtypes of RGCs have been identified to date. Here, we analyze 6225 RGCs (average of 5000 genes per cell) from right and left eyes by single-cell RNA-seq and classify them into 40 subtypes using clustering algorithms. We identify additional subtypes and markers, as well as transcription factors predicted to cooperate in specifying RGC subtypes. Zic1, a marker of the right eye-enriched subtype, is validated by immunostaining in situ. Runx1 and Fst, the markers of other subtypes, are validated in purified RGCs by fluorescent in situ hybridization (FISH) and immunostaining. We show the extent of gene expression variability needed for subtype segregation, and we show a hierarchy in diversification from a cell-type population to subtypes. Finally, we present a website for comparing the gene expression of RGC subtypes.

BackgroundAs obligate blood-feeding arthropods, ticks transmit pathogens to humans and domestic animals more often than other arthropod vectors. Livestock farming plays a vital role in the rural economy of Pakistan, and tick infestation causes serious problems with it. However, research on tick species diversity and tick-borne pathogens has rarely been conducted in Pakistan. In this study, a systematic investigation of the tick species infesting livestock in different ecological regions of Pakistan was conducted to determine the microbiome and pathobiome diversity in the indigenous ticks.Methodology/Principal findingsA total of 3,866 tick specimens were morphologically identified as 19 different tick species representing three important hard ticks, Rhipicephalus, Haemaphysalis and Hyalomma, and two soft ticks, Ornithodorus and Argas. The bacterial diversity across these tick species was assessed by bacterial 16S rRNA gene sequencing using a 454-sequencing platform on 10 of the different tick species infesting livestock. The notable genera detected include Ralstonia, Clostridium, Staphylococcus, Rickettsia, Lactococcus, Lactobacillus, Corynebacterium, Enterobacter, and Enterococcus. A survey of Spotted fever group rickettsia from 514 samples from the 13 different tick species generated rickettsial-specific amplicons in 10% (54) of total ticks tested. Only three tick species Rhipicephalus microplus, Hyalomma anatolicum, and H. dromedarii had evidence of infection with “Candidatus Rickettsia amblyommii” a result further verified using a rompB gene-specific quantitative PCR (qPCR) assay. The Hyalomma ticks also tested positive for the piroplasm, Theileria annulata, using a qPCR assay.Conclusions/SignificanceThis study provides information about tick diversity in Pakistan, and pathogenic bacteria in different tick species. Our results showed evidence for Candidatus R. amblyommii infection in Rhipicephalus microplus, H. anatolicum, and H. dromedarii ticks, which also carried T. annulata.

A cross-sectional study was carried out to determine the prevalence, species characterization, and associated risk factors with Eimeria of cattle of district Toba Tek Singh from April, 2009 to March, 2010. Of the total 584 fecal samples examined for Eimeria, 275 (47.09%) were found infected with six species of Eimeria. Among the identified species of Eimeria, Eimeria bovis was found to be the highest prevalent species (52.36%), followed in order by Eimeria zuernii, Eimeria canadensis, Eimeria ellipsoidalis, Eimeria alabamensis, and Eimeria cylindrica with prevalence of 48.27%, 34.83%, 29.31%, 24.14%, and 8.62% respectively. Peak prevalence was observed in August. Cattle were infected more frequently during rainy (60.32%) and post-rainy seasons (59.25%). Calves had significantly higher prevalence (P<0.05) of Eimeria than adults while higher prevalence of Eimeria was observed in female cattle. Among management and husbandry practices, feeding system, watering system, housing system, floor type, and herd size strongly influenced the prevalence of Eimeria in cattle. Coccidiosis was more prevalent in ground feeding system, pond-watered animals, closed housing system, and non-cemented floor type (P<0.05) as compared to trough feeding system, tap watered animals, open housing system, and partially cemented floor types, respectively. Breed and body condition of animals were not found risk factors (P>0.05) influencing prevalence of Eimeria.

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The objective of the present study was to determine the prevalence and associated determinants (e.g., sex, age, on-farm management and husbandry) of gastrointestinal (GI) helminths in the domestic animals of district Toba Tek Singh, Punjab, Pakistan. For this purpose, 1,140 cattle, 1,140 buffaloes, 660 goats, 840 sheep, and 156 camels were randomly selected and their fecal samples were screened every other week for a year using a modified floatation technique. The samples positive for strongyle-type eggs had the parasite species identified using coproculture. It was found that the prevalence of GI helminths was significantly higher (P < 0.05) in sheep (44.17%; 371/840) than in other livestock. Sheep were followed in order by goats (40.15%; 265/660), buffaloes (39.82%; 454/1,140), and cattle (33.68%; 384/1,140). The important helminth species identified were Fasciola (F.) gigantica, Fasciola hepatica, Haemonchus contortus, Toxocara vitulorum, Trichostrongylus spp., Oesophagostomum spp., Ostertagia spp., Cooperia spp., Strongyloides spp., Moniezia spp., and Trichuris spp. The prevalence of GI helminths except F. hepatica and F. gigantica was significantly higher in grazing animals, females (P < 0.05) and young (P < 0.05) of all the host species when compared with stall-fed animals, males and adults, respectively. Using ponds and rivers/canals as drinking water were found to have significant influence (P < 0.05) on the prevalence of GI helminths. The results provide a baseline data for planning future research and control strategies against GI helminthes.