Glaucomatous optic neuropathy (GON) is an anatomofunctional impairment of the optic nerve triggered by glaucoma. Recently, growth factors (GFs) have been shown to produce retinal neuroenhancement. The suprachoroidal autograft of mesenchymal stem cells (MSCs) by the Limoli retinal restoration technique (LRRT) has proven to achieve retinal neuroenhancement by producing GF directly into the choroidal space. This retrospectively registered clinical study investigated the visual function changes in patients with GON treated with LRRT.Twenty-five patients (35 eyes) with GON in progressive disease conditions were included in the study. Each patient underwent a comprehensive ocular examination, including the analysis of best corrected visual acuity (BCVA) for far and near visus, sensitivity by Maia microperimetry, and the study of the spectral domain-optical coherence tomography (SD-OCT). The patients were divided into two groups: a control group, consisting of 21 eyes , and an LRRT group, consisting of 14 eyes .p<0.05), whereas the mean increases were not statistically significant in controls (p>0.5).After 6 months, the BCVA, close-up visus, and microperimetric sensitivity significantly improved in the LRRT-treated group  both in BCVA and sensitivity and an improvement of residual close-up visus, in the comparison between the LRRT results and the control group. Further studies will be needed to establish the actual significance of the reported findings. Glaucoma causes retinal ganglion cell (RGC) layer degeneration, impairing visual function. It has been estimated that over 100 million people will be affected by glaucoma in 2040 . The damNumerous pathogenetic mechanisms have been proposed to explain the apoptotic loss of RGCs in glaucoma: microcirculatory ischemia, hyperproduction of reactive oxygen species (ROS), impaired bidirectional axonal transport, parainflammation, excitotoxicity, neurotrophic factors reduction, and electrical activity reduction –5. CurreThe recent appearance of cell therapy in regenerative medicine has represented a promising tool in glaucoma therapy . On the On the other hand, mesenchymal stem cells (MSCs) can be exploited for their paracrine secretion of different molecules that have been shown to activate RGC-intrinsic regenerative programs after optic nerve injury, promoting cell survival and axonal regeneration . SpecifiIn this context, an autograft of MSCs in the suprachoroidal space according to the Limoli retinal restoration technique (LRRT), consisting of the following triad, could be effective: adipose stromal cells (ASCs), adipose-derived stem cells (ADSCs) contained in the stromal vascular fraction (SVF) of adipose tissue, and platelets (PLTs) recovered from the platelet-rich plasma (PRP) –13 . Written informed consent was obtained from all the patients prior to enrollment.Furthermore, patients with minor damage preferred clinical follow-up without surgery because they did not feel the damage progression, whereas treated patients chose surgery as they felt that damage progression, despite the good tonometric control. Many patients included in the study were pseudophakic, and the ones still phakic did not have a significant cataract that could interfere with vision. Individuals who met the following inclusion criteria were recruited for the study.Excavated optic papilla with cup/disc <0.6.Good therapeutic balance (IOP ≤15 mmHg) obtained with hypotonizing therapy; the pressure must be corrected also after LRRT surgery.Best corrected visual acuity (BCVA) between +1 and 0 logarithm of the minimum angle of resolution (LogMAR).Age between 50 and 84 years old.Stable eye conditions without previous surgery for ocular trauma.Diagnosis of GON highlighted by microperimetry or, when necessary, coherence optical tomography (OCT):Bad therapeutic balance (IOP > 15 mmHg) obtained with hypotonizing therapy; IOP must be also corrected after LRRT surgery.Refractive error ≥ 6 diopters of myopia, hypermetropia, and astigmatism.Close-up visus not evaluable.Presence of cataract or other media opacity that could interfere with a functional response.Presence of chorioretinal diseases including macular pucker with the altered foveal area, age-related macular diseases (AMD), or eredodistrophy.Intravitreal injection treatment and/or intraocular surgery.Inability to provide written informed consent.Inability to attend all follow-up visits.Systemic diseases including multiple sclerosis, epilepsy, vasculitis, Parkinson’s disease, renal and hepatic diseases, malignant neoplasms, and decompensated diabetes mellitus.Subjects who met any of the following criteria were excluded from the study:Some patients presented indications for bilateral treatment , and other cases presented indications for unilateral treatment . Or according to the patient’s consent.All the eyes enrolled in this study were divided into two groups: the LRRT group who underwent autologous suprachoroidal graft of mesenchymal cells and the control group of GON patients who did not undergo LRRT surgery. Participants used as control were matched with GON patients according to the sensitivity alteration measured by microperimetry.The diagnosis of GON was established for each patient by the clinical analysis of visual performances (VP). Then, evaluation by slit-lamp biomicroscopy with and without dilatation, applanation tonometry, and retinal mapping with an indirect ophthalmoscope was performed. Also, BCVA, close-up visus, sensitivity measured by microperimetry (MY) with Maia 100809 , spectral domain-optical coherence tomography (SD-OCT) with Cirrus 5000 , and ocular electrophysiology with the Retimax electromedical system  were performed. All the ophthalmologic analyses were carried out by the same examiner at baseline (T0) and 6 months (T180) in both groups. Finally, the subjective improvement of VP in the LRRT group at 6 months after surgery was reported. BCVA was always measured according to the standards recommended by the early treatment diabetic retinopathy study charts (ETDRS) at 4 m and expressed in logMAR. The visual acuity for near distance (close-up visus) was recorded in points (pts). Microperimetry was performed using a Maia apparatus  with images acquired by scanning laser ophthalmoscopy. The perimetry can indicate only an existing damage, and its results are not always reproducible, whereas the micro-perimetry provides more reproducible and reliable results due to the eye-tracker function on the fixation. Furthermore, it provides information on fixation whose stability is a positive parameter after visual rehabilitation. Sensitivity was measured from 0 to 25 dB, and the color was coded. The field of the infrared image was 36° × 36°, and perimetry was performed in a field of 30° × 30° with a luminance of 4 asb. The full-threshold 4-L test was used to assess the retina in detail.The autograft of MCs in the suprachoroidal space, i.e., LRRT, consisted of the following triad: ASCs, ADSCs contained in SVF of adipose tissue, and platelets obtained from the PRP. ASCs were collected from the orbital fat during the surgical procedure according to previously published methods . A sclerg for 5 min at 20°C in order to isolate SVF from the mature adipocytes, connective tissues, cellular debris, and oil.After exposing the choroid, the pedicle of adipose tissue derived from the orbital space was placed on the choroid’s surface Fig. . ADSCs cg for 5 min at 20° C in order to isolate the PRP.The platelets were obtained from PRP gel according to the established methodologies . Eight mThe adipose pedicle was infiltrated with platelets derived from the PRP gel Fig. .Finally, a mixture of ADSCs from the SVF and PRP was used to saturate the residual volume of the scleral pocket, where the pedicle of adipose tissue-derived from the orbital space was previously placed Table .Table 1g to collect the cellular SVF as a pellet. Once isolated, characterization of the cell composition of freshly isolated SVF was obtained by multi-color flow cytometry  that allows the in vitro identification of the surface marker expression of the cells. The panel of cell surface antigens was chosen in agreement with the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT) recommendations [Flow cytometry analyses were performed in order to identify the phenotypic characteristics of the population of cells within the graft, specifically ADSCs and platelets. PRP and SVF were obtained from patients of the LRRT group who underwent LRRT surgery and were isolated under fresh conditions. SVF was manually isolated from each patient’s lipoaspirate in a clean room near the operating room, according to a previously described method . Brieflyndations . The immndations .t test was used to compare the values between controls and the study group. The paired t test was run to compare the study subjects and controls at baseline and after 6 months. A p value < 0.05 was considered statistically significant.All statistical analyses were performed using the software SPSS Statistics . Data were summarized with the mean ± standard deviation (SD), and minimum and maximum (min-max) values were also reported. Student’s A total of 35 eyes from 25 patients affected by GON  met the inclusion criteria and were enrolled in the study. Twenty-one eyes of the total composed the control group , while the remaining 14 eyes constituted the LRRT group of patients . Two patients from the latter group were excluded from the analysis because of the close-up visus that was not evaluable and the BCVA that was greater than 1 LogMAR. The baseline characteristics are summarized in Table No adverse event associated with the surgery either intra-operatively or post-operatively was observed throughout the period. The mean values of the IOP recorded before and after surgery did not change significantly. In the control group, the mean IOP in T0 was 13.76 mmHg (range 10–15 ± 1.64 SD), and in T180, 13.33 mmHg (range 10–15 ± 1.39 SD). In the LRRT group, the mean IOP in T0 was 13.29 mmHg (range 10–15 ± 1.63 SD), and in T180, 13.29 mmHg (range 10–15 ± 1.59 SD). All completed 6 months of evaluation.5 cells.The positive expression for cell surface antigens CD61 and CD41 identified PLTs. We observed that the PRP contained a mean of 79.2 ± 13.7% PLT on a total of 105 cells and the ADSC/μL was 590.3/μL (127.2–1485.3/μL)  and from 0.213 to 0.155 logMAR in the LRRT group with a statistically significant increase of −0.0582  .After 6 months, the BCVA went from 0.0947 to 0.0937 logMAR in the control group with a mean increase of −0.001  and from 10.21 to 8.29 pts in the LRRT-treated group with a mean increase of 1.93  .After 6 months, the close-up visus went from 6.57 to 6.9 pts in the control group with a mean reduction of −0.33 pts  Fig. , Table 3p > 0.05) and from 10.00 to 11.12 in the LRRT-treated group with a statistically significant increase of 1.12 dB  .After 6 months, sensitivity went from 13.20 to 12.64 dB in the control group with a mean reduction of −0.56 dB  and remained unvaried in 3 eyes (21%). Notably, the VP worsened in no eyes (Fig. 9% and reIn this study, patients affected with GON received the LRRT treatment to preserve the residual VP. All of the eyes in the study group showed improvement in BVCA, sensitivity, and residual close-up visus with no ocular and systemic complications, whereas the majority of the eyes in the control group showed a decrease in the same measured parameters. The improvements were consistent through the 6-month follow-up. Furthermore, we monitored IOP throughout the study and included patients affected with GON whose IOP was well within normal parameters. There were no variations in our case study.GON is currently recognized as a progressive neurodegenerative disease, resulting in due course permanent visual loss . Up to dThese cells are ubiquitously distributed in the body and play a key role in organogenesis, tissue remodeling, and repair . A growiThey are characterized by multipotency, as they have been shown to differentiate into several cell types, including adipocytes, chondrocytes, osteoblasts, vascular endothelial cells, cardiomyocytes, beta-pancreatic cells, hepatocytes, and, under specific conditions, also into retinal neuron-like, including retinal progenitors and photoreceptors . HoweverThese biological mediators are well known for providing a vital microenvironment by inducing gene expression changes that lead to neuroprotective, regenerative, anti-inflammatory, and anti-apoptotic effects; hence, they can support cell survival and rescue the damaged tissue –8, 18. TMoreover, growth factors secreted by the grafted cells in cell therapy could influence the autophagic processes. Autophagic markers increase in optic nerve damage conditions such as glaucoma. In fact, ultrastructural and biochemical results have shown that autophagy is significantly activated in RGCs after chronic IOP elevation. Then, it reduces after a few weeks . The useAs defined by the International Society for Cellular Therapy, MSCs are characterized by a set of negative and positive surface markers; they express CD105, CD73, CD34, and CD90 and lack CD45, CD14, CD11, CD79, CD19, and HLA-DR. MSCs also express other surface markers, such as CD44, CD166, Stro-1, CD106, and CD146 [MSCs can be isolated from adult and fetal tissues, including bone marrow, adipose tissue, Wharton’s jelly, dental pulp, and placenta , 28. ADSThe LRRT is a cell therapy consisting of autologous ADSCs within the SVF, ASCs, and PRP ; it is aAlongside ADSCs, also ASCs have shown regenerative potential as well as autologous PRP that is a source of growth factors , 28. FreWe want to remind that some ways different of administration of MSCs have been explored in several clinical studies for the management of degenerative retinal diseases. We used the suprachoroidal method, which is reported to have no serious complications and is considered to be safer compared to the intravitreal or subretinal applications .The choice to use the suprachoroidal route, rather than other forms of administration, is determined by clinical safety as well as by good efficacy. The sub-tenonian route has been used with discrete efficacy and absolute safety to graft stem cells  and PRP 2, thus representing a minimal space of the whole bulbar surface. Also, in LRRT surgery, there is no compression of the supra-choroidal space as the scleral flap under which is situated the cell graft is gently sewn. This is very important because if the cell graft is compressed, the adipose pedicle inserted could become ischemic and atrophic. The GFs secreted by the ADSCs can effectively pass through that space and reach the retinal target without producing immune reactions, making the suprachoroidal region ideal as the site of MSC administration.The suprachoroidal area has been shown as a natural drug storage and an immune-protected region –13. The The effect of ADSCs is thought to be related to the expression of several GFs, including bFGF, BDNF, NGF, CNTF, GDNF, and HGF , 6, 7. GIn this way, it can promote RGC survival and stimulate both axonal regeneration and myelination in the optic nerve, restoring both dendritic and synaptic connections with bipolar and amacrine cells –36.According to these findings, MSCs might promote RGC function and survival through the paracrine release of GFs, exosomes, and microvesicles over time, slowing retinal degeneration.These biochemical mechanisms could underlie the positive clinical results we observed following the autologous MSC graft performed by LRRT treatment in the suprachoroidal space in patients affected with GON.The LRRT treatment has been applied in other studies of our group in retinal diseases, such as retinitis pigmentosa, AMD, and optic neuropathies, and its safety and effectiveness with improvements of both VP and electroretinographic parameters has been shown –12. In tIn accordance with our results, many investigators evaluated the safety and efficacy of the MSC use for retinal diseases and suggest MSC-mediated neuroprotection , 39, 40.Oner et al.  showed tIn Brazil, Siqueira et al.  conducteFinally, a Californian group  obtainedSecond, the duration of action of LRRT treatment is unknown. Even though long-term research is necessary to determine the duration of efficacy, PRP booster injections after 12 months have been shown to maintain the outcomes. Another limitation of the study is that we do not measure whether additional treatments such as electrical stimulation may increase MSC activity. The latter limitation forms the basis for near-future studies.The LRRT surgery has proven safe and effective in treating patients affected with GON.In our experience, both visual acuity and retinal sensitivity measurements showed statistically significant improvements in 80% of GON patients after LRRT during the follow-up period of 6 months, and no ocular or systemic effects were reported. Therefore, autologous MSC graft combined with PRP into the suprachoroidal space could contribute to restoring optic nerve function, improving the clinical, prognostic, and rehabilitative aspects in patients affected with GON that currently have no curative treatment options.Further studies are needed to validate our findings and to unveil the potential of MSCs as therapeutic agents in regenerative medicine especially for degenerative retinal and optic nerve diseases.


South American camelids (SACs) constitute the greatest livestock wealth of the Andean populations. Approximately half a million people from the high Andean areas are dedicated to the breeding of SACs as their main activity. In general, infectious diseases, particularly diarrheal infections, cause high morbidity and mortality in offspring and adult animals. In the study, we demonstrated that multiple virus pathogens circulate among neonatal SACs, and coinfections from other viruses might be common among SAC crias. We also demonstrated, for the first-time anywhere, the circulation of mammalian orthoreovirus in SACs or camelids. Diarrheal infections can potentially impact livestock productivity, which translates into serious economic losses for the Peruvian SAC industry, especially within rural communities, directly impacting their livelihood. Better knowledge of the infections that afflict these animals will enable the implementation of measures for the prevention and control of pathogens, the results of which will ultimately be reflected in improving the quality of life of these communities.Enteric infections are a major cause of neonatal death in South American camelids (SACs). The aim of this study was to determine the prevalence of enteric viral pathogens among alpacas and llamas in Canchis, Cuzco, located in the southern Peruvian highland. Fecal samples were obtained from 80 neonatal alpacas and llamas and tested for coronavirus (CoV), mammalian orthoreovirus (MRV), and rotavirus A (RVA) by RT-PCR. Of the 80 fecal samples analyzed, 76 (95%) were positive for at least one of the viruses tested. Overall, the frequencies of positive samples were 94.1% and 100% among alpacas and llamas, respectively. Of the positive samples, 33 (43.4%) were monoinfected, while 43 (56.6%) had coinfections with two (83.7%) or three (16.3%) viruses. CoV was the most commonly detected virus (87.5%) followed by MRV (50%). RVA was detected only in coinfections. To our knowledge, this is the first description of MRV circulation in SACs or camelids anywhere. These data show that multiple viruses circulate widely among young alpaca and llama crias within the studied areas. These infections can potentially reduce livestock productivity, which translates into serious economic losses for rural communities, directly impacting their livelihoods. Cryptosporidium spp., Giardia spp., Salmonella spp., Escherichia coli, Clostridium spp., rotavirus, and coronavirus [South American camelids (SACs) constitute the greatest livestock wealth of Andean populations. The raising of alpacas and llamas is a crucial economic activity in Andean regions, enabling the production of fibers; primarily those of alpacas, which have a high value in international markets because of their fine texture. The llama, due to its size and physical strength, is also used as a pack animal and plays an important role in rural transportation. Peru possesses three million alpacas, the largest alpaca population in the world, and has a herd of approximately one million llamas. Most of these animals are found in the departments of the southern highlands, particularly in Puno and Cuzco. Over 80% of Peru’s alpacas and the entire population of llamas are kept by small producers who lack adequate infrastructure for conducting both production and marketing activities of their products ,2,3. Acconavirus ,10,11. Despite the vital importance of alpaca and llama breeding to the Peruvian economy, studies on enteric infections, particularly viral infections, in SACs are incipient. A better understanding of the epidemiology of enteric infections is essential to develop preventive measures. The aim of this study was to determine the prevalence of enteric viral pathogens—specifically coronavirus (CoV), mammalian orthoreovirus (MRV), and rotavirus A (RVA)—associated with infections among alpacas and llamas in Canchis, Cuzco, located in the southern Peruvian highland.CoV and RVA have already been identified as important diarrheal pathogens in neonatal SACs. On the other hand, MRV has not been associated to enteric infection. However, in a previous study, during the characterization of RVA strains detected in feces of alpacas and llamas collected in February 2014 in the province of Canchis in the state of Cusco, Peru, three viral strains containing 10 segments of double-stranded RNA (dsRNA) suggestive of MRV were isolated in an MA-104 cell culture . In thishttps://www.ncbi.nlm.nih.gov/nucleotide/, accessed on 5 April 2021). Sequences generated in this study were deposited in GenBank under the accession numbers MN200219, MN200220, and MN200221.The protocol used for isolating MRV in cultures of African green monkey cells (MA-104) has been described elsewhere . Viral cn = 68) and llamas (n = 12), all one to five weeks old, with  and without  diarrhea, from three camelid breeding areas of the districts of Marangani (Silly and Quisini) and Sicuani , province of Canchis, department of Cuzco, Peru. Samples were collected during the birthing season of alpacas and llamas, in January and February 2015. Fecal samples were collected directly from the rectum of animals and kept at −20 °C until processing at the Laboratory of Veterinary Virology and Immunology, Universidad Nacional Mayor de San Marcos, Lima, Peru.Eighty fecal samples were collected from neonatal alpacas  according to the manufacturer’s recommendations.The extraction of viral RNA samples required an initial clarification process. Fecal suspensions were prepared at 10–20% in PBS (pH 7.2) and then centrifuged at 2500× ® Green Master Mix , respectively. The genomic RNAs were subject to one cycle of reverse transcription (5 min at 25 °C followed by 45 min at 42 °C), and one step of 2 min at 95 °C followed the cycles of PCR. Samples were submitted to RT-PCR and nested PCR using specific primers . ReverseEmbecovirus ), using the first-round PCR products and specific CV2U and CV2L primers [For CoV testing, the primers Cor-FW and Cor-RV, which amplify a 251-bp fragment of the RNA-dependent RNA polymerase (RdRp) gene of any CoV, were used for the first round of PCR, according to the protocol described previously . Five mi primers  that ampMRV detection was performed by one round of RT-PCR using the primers MRV-FM and MRV-RM. The primers used for MRV RT-PCR detection target the conserved genomic segment that encodes the RdRp. Selection was carried out in silico by aligning all MRV genomes deposited on GenBank, representatives of all serotypes (MRV1-MRV4), with a predicted product of 181 bp. The specificity of the primers was tested by amplifying the cell-cultured MRV strains and confirming the results by sequencing analysis. Sequences generated from the RdRp 181-bp amplicons were also deposited in GenBank under the accession numbers MN200216, MN200217, and MN200218. The PCR conditions were as follows: 3 min at 95 °C; followed by 40 cycles of PCR, each consisting of 40 s at 94 °C, 1 min at 45 °C, and 1 min at 72 °C; and the final extension step for 5 min at 72 °C. RVA was detected using primers that amplify the NSP5-encoding gene, as described previously ,16. w/v) agarose gel electrophoresis, stained with ethidium bromide, and visualized under UV light. A 100-bp DNA ladder  was used to determine molecular size.All primers were synthesized by IDT . Reactions were performed in a Bio-Rad T100 PCR thermal cycler . The PCR products were separated by 1.5% , identified as MRV-1, were used. RV strains RVA/Alpaca-tc/Per/ SA44/2014/G3P(30) (GenBank accession number KT935485) and RVA/Human-wt/Per/FRNM/2014/G3P(8)(14)(40)(50) (GenBank accession number KY972105), belonging to rotavirus species A (RVA), were used as positive controls.To validate the PCR assays, positive controls were used for each of the studied viruses, which consisted of viral strains isolated in cell culture whose identification was previously confirmed by sequencing analysis. Strains AlpCoV-SA44 and AlpCoV-HN , both BetaCoV, subgenus Analysis of the migration profile of the dsRNA genome of three viral strains  isolated in MA-104 cells revealed an electrophoretic migration profile compatible with MRV . A sequep = 0.1048). Specifically, among the 68 samples from alpacas, 26 were from animals with diarrhea and all tested positive. Of the 42 samples from alpacas without diarrhea, 38 tested positive. Among the 12 samples from llamas, 9 and 3 were from animals with and without diarrhea, respectively, and all tested positive.Of the 80 fecal samples, 76 (95%) were positive for at least one of the screened viruses. Overall, the frequencies of positive samples were 94.1% and 100% among alpacas and llamas, respectively . Positivn = 29) of monoinfections and 95.3% (n = 41) of coinfections. MRV was found in 12.1% (n = 4) of monoinfections and 83.7% (n = 36) of coinfections. RVA was only found in coinfections   were monoinfected, while 43 (56.6%) had coinfections of two or three viruses . The com n = 16) .Embecovirus, with the majority of these samples (15/16) obtained from alpacas. The genus was not identified for 4 (5.7%) and 71.4% of CoV and BetaCoV samples, respectively  were identified as BetaCoV. Of these, 16 (22.9%) belonged to the subgenus ectively .There are few reports on enteric viral infections in SACs, particularly in Peru ,10,17,18In Peru, the SAC breeding area is geographically located over 3800 m above sea level. This presents an extreme climate and poor forage for animal species that are not typical of the Andean plateau, although it is possible to observe the rare presence of traditional livestock . An estimated one and a half million people from the high Andean areas were engaged in the breeding of SACs as their primary economic activity in the year 2000. However, the per capita income in these camelid-producing areas is the lowest in the country ,20.The economic conditions of peasant communities such as those studied here, which are dedicated to the breeding of SACs, are chaotic due to extreme poverty. Their situation is becoming even more critical due to the lack of drinking water, vital hygienic services, and healthcare. These shortages are reflected in the health of their animals, which suffer greater disease burdens due to inadequate handling and lack of prophylactic programs. The animals are managed in extensive systems that use swampy areas watered by streams or rivers. Infectious and parasitic diseases are major limiting factors in the production of these animals. In general, infectious diseases cause high morbidity and mortality in both offspring and adult animals, which translate into serious economic losses for rural communities, directly impacting their livelihood ,6,21.Our data showed a wide circulation of the surveyed viruses among alpacas and llamas in the three studied communities. CoV and RVA have already been identified as important diarrheal pathogens in neonatal SACs ,10,17,22Previous studies conducted in the Andean areas of the Peruvian highlands have shown the circulation of CoV in alpacas on farms with semi-intensive systems and in communities with extensive systems ,8,22, wiBetacoronavirus genus, do not belong to the Embecovirus subgenus. These data suggest the circulation of different genera and subgenera of CoV among these animals.Nucleotide sequences of alpaca CoV strains exhibited high identity with circulating bovine strains , suggesting a possible bovine origin of these viruses ,16,30,31RVA has already been associated with outbreaks of diarrhea among alpacas and llamas in the Peruvian highlands with high mortality rates ,9,22,32.Virus-positive samples were detected among animals with and without diarrhea. The occurrence of asymptomatic infections in these animals could be due to the persistence of maternal immunity, or to the fact that as these pathogens are circulating widely in the herd, animals could have been infected before the study period and may have developed immunity, thus not presenting with diarrhea. Asymptomatic shedding is a source of contamination favoring the environmental persistence of these pathogens.One of the most impressive findings of this study was the extremely high prevalence of coinfections 56.6%). This can be explained by (i) the high sensitivity of the detection technique used in the study, (ii) the high circulation of these viruses among herds, and (iii) inadequate sanitary conditions for managing the animals, allowing the dissemination of pathogens. Unfortunately, we do not have information that allows us to infer the impact of coinfections on clinical presentations. However, it has been demonstrated that coinfections probably increase the severity of diarrhea in alpacas [.6%. ThisThe raising of alpacas and llamas in Peru is conducted in a particular ecological niche by rural communities and is characterized by the close contact of shepherds with animals as well as failures in sanitary and prophylactic programs due to lack of economic resources. These factors facilitate interspecies transmission of viruses such as CoV, MRV, and RVA, triggering possible zoonotic or anthropozoonotic infections ,33,34. C


Most endocervical adenocarcinomas (EAC) are associated with high-risk HPV (hrHPV) infection, with HPV genotypes 16, 18 and 45 accounting for >90% of the cases. Among endocervical glandular lesions, screening with hrHPV test has previously shown to predict the outcome better than cytology, although around one-fifth of the EAC remain negative both in hrHPV testing and cytology. The study consists of two consecutive HPV-primary screening rounds, conducted in 2012–2015 and 2017–2020. Of the 87 women aged 35 to 60 years of age diagnosed with Atypical endocervical cells, NOS or Atypical endocervical cells, favor neoplastic cytology during the first screening round, 63 (72.4%) were hrHPV positive and 24 (27.6%) were hrHPV negative. Among hrHPV positive patients, three EAC, two adenocarcinomas in situ (AIS), one AIS + high-grade intraepithelial lesion (HSIL) and 13 HSIL were found. Of the histologically verified lesions, 68.4% (13/19) were purely of squamous origin. All the EAC and AIS were HPV16 or HPV 18 positive. No high-grade histological lesions were found among the hrHPV negative patients with cytological glandular atypia. A later database search revealed one HPV-negative, gastric-type mucinous EAC that was missed by the HPV primary screening. In Finland, national cervical cancer screening has been organized since the 1960s. Women from the age of 30 to the age of 60 are invited to participate every fifth year, and in some municipalities 25- and 65-year-olds are also included. Since the beginning of the organized screening, the number of cervical cancer deaths in Finland has decreased to one-fifth of its original number [Traditionally, the conventional Pap smear has been the primary test. In 2012, the city of Tampere, and later all municipalities in Pirkanmaa region, started HPV primary screening with high-risk HPV (hrHPV) test and cytological smear as a triage in women aged ≥35 years ,5,6.The important role of hrHPV infection leading to the development of invasive cervical squamous cell carcinoma (SCC) and behind the most of the endocervical adenocarcinomas (EAC) has been well documented ,8,9,10. In the previous studies, the clear cell adenocarcinomas (AC) accounted for 3.9% to 4%, the serous AC for 3% to 3.2%, the endometrioid AC for 1.4% to 3%, the minimal deviation AC 1.6% to 6% and the AC, not otherwise specified, for 4.7% to 8% of the EAC . These lIn previous studies, HPV testing identified earlier more women with cancer or precancerous lesions than cytology alone ,14,15,16The aims of the present study are to evaluate the detection and outcome of endocervical atypia in an HPV primary screening programme.The study cohort represents women participating in an HPV primary screening programme organized by Fimlab Laboratories during 2012–2015 in the Pirkanmaa region, Finland. During these years, altogether 93.439 women aged 35 to 60 years were invited to cervical cancer screening, which included both the hrHPV test and the conventional pap test. All the pap tests of the hrHPV positive women were analyzed and, as a quality assurance, 10% of the HPV negative patients were assessed cytologically in the 2012–2016 period.In total, 66.147 (70.8%) of the invited women participated in the screening. Out of the participants, 87 (0.13%) were diagnosed with endocervical glandular atypia  with or without a squamous atypia .At the beginning of the study, according to the screening protocol and the national guidelines, women with a positive hrHPV test result and/or a cytological diagnosis of Atypical Squamous Cells, Undetermined Significance (ASC-US) and/or Atypical Endocervical Cells, NOS  were referred to repeat sampling after 12 months . The repIn 2016, there was a change in the national guidelines after which patients with cytological diagnosis of AEC, NOS with or without a positive hrHPV test result were referred for a colposcopy immediately.Women with a cytological diagnosis of Atypical Endocervical Cells, Favor Neoplastic  and/or a squamous cytological diagnosis of Low-Grade Squamous Intraepithelial Lesion (LSIL) or worse were always immediately referred for a colposcopy despite the hrHPV status of the patient. After the colposcopy, the patient was treated according to the national guidelines . DespiteAt the first screening round in 2012–2015, the patients were between 35 to 60 years old, and at the second screening round in 2017–2020, they were between the ages of 40 to 65 years.All diagnoses on cervical cytological samples were provided according to the Bethesda Classification for Reporting Cervical Cytology 2014 ,19,20. TThe HPV status, genotype, cytological and histological data were retrieved from the Laboratory Information System (LIS) of the Fimlab Laboratories Oy. Later, an additional LIS- search was conducted to find possible EACs missed by the primary HPV screening during the study period 2012 to 2020. For the statistical analysis, SPSS version 25 was used .Because the samples of the study include those produced by the national cervical cancer screening protocol and its follow-ups, the individual consent of each participant was not requested. The study was approved by the Ethical committee of Pirkanmaa Health Care District (R13094 and R16022). The study was conducted according to the Declaration of Helsinki.After the first screening rounds in 2012–2015, 61 patients were diagnosed as AEC, NOS on cytology . Of thosIn 2012–2015 a total of 17 patients were diagnosed with AEC, NOS in combination with a squamous cytological diagnosis . Out of A cytological diagnosis AEC, FN was provided seven times in the first screening rounds in 2012–2015 and AEC, FN + LSIL was diagnosed two times . All patAltogether, behind the 87 cytological endocervical glandular diagnoses with or without combined squamous cytological atypia, one EAC, two AIS, two EAC + LSIL, one AIS + HSIL and 13 HSIL were found reflecting that 68.4% (13/19) of the high-grade lesions verified histologically were purely of squamous origin . There ap-value > 0.05) to endocervical malignancies or to squamous neoplasias.Of the endocervical malignancies, 33.3% (2/6) were HPV18 positive and 66.7% (4/6) were HPV 16 positive . Among HAside for the two above-described AIS cases with the wide time lag before the histological diagnosis, all the high-grade histological lesions were diagnosed during the first screening round. No high-grade histological lesions were found among patients with cytological endocervical glandular atypia and a negative hrHPV test result , Table 2The diagnosis of this gastric-type mucinous EAC was made two years after the negative HPV primary screening result. The diagnosis was immunohistochemically confirmed, and the patient was treated with hysterectomy, combined with salpingo-oophorectomy and lymphadenectomy. There were no metastases. The tumor itself was not tested for hrHPV, but the vaginal hrHPV tests have remained negative since. A 5-year follow-up has been conducted, and no additional tumors were observed.During the first screening round, 17.5% (11/63) of the hrHPV positive patients and 29.2% (7/24) of the hrHPV negative patients left follow-ups unattended . Of the The results of the present study are in agreement with the previously published series in the sense that no endocervical glandular malignancies were found among patients with endocervical cell atypia and a negative hrHPV result, reflecting that the majority of the endocervical adenocarcinomas are associated with hrHPV infection ,9,10. InIn addition to the lack of glandular malignancies, in the present study, there were no histological HSIL or SCC among the cases with endocervical glandular atypia and a negative hrHPV result. This is supporting the previous findings, according to which over 60% of the mild cytological glandular atypias with or without mild squamous atypia represent benign changes, or low-grade squamous histological lesions ,27,28,29In our study, behind the cytological endocervical glandular diagnoses, with or without combined squamous cytological atypia, 68.4% of the clinically significant lesions  were purely of squamous origin. This might be caused by the extension of HSIL to endocervical glands, which is known to be a common cause of false cytological glandular diagnoses, or simply the result of the everyday struggle pathologists have in separating the cytological glandular features from squamous features ,26,30. NIn previous studies, the hrHPV test has been more often positive for cancer and precancerous lesions than cytology, and the difference in performance has been more pronounced among cases subsequently diagnosed with AIS or EAC than CIN3 or SCC ,12,15,16Similarly, in the present study, only two malignancies were found during the second screening rounds. Both lesions represented AIS, and their histological diagnoses were not reached until 5 years and 9 months and 6 years and 11 months after the initial hrHPV positivity and cytological glandular atypia at the study baseline. Although the participation rate for screening (70.8%) in the present study was similar to that previously reported in Finland , the higOur database search revealed one hrHPV-negative mucinous, gastric type adenocarcinoma case missed by the primary HPV screening during the study years 2014–2021. Since the hrHPV test at the screening was negative, no follow-ups were scheduled for the patient. The cancer was later diagnosed on a gynecological check-up for other reasons.Since, in previous studies, 22% of the EAC and around 15% of the AIS were negative both in cytology and in hrHPV test, it can be only speculated if including the cytological sample to the screening protocol would have led to an earlier diagnosis in this case ,12. It hIn our series, all AIS and EAC were positive with either HPV16 or HPV18. Out of the HSIL, 42.9% were positive with an hrHPV type other than HPV16 or HPV18. In the statistical analyses, the HPV types  or combinations of them did not provide a prediction specifically relating to endocervical malignancies or to squamous lesions. This may have been due to the small number of positive cases, which may also have been the reason why the positive cases were not analysed further.In conclusion, in this study of two consecutive HPV-primary screening rounds, among 87 women diagnosed with AEC, NOS or AEC, FN in cytology, three EAC, two AIS, one AIS + HSIL and 13 HSIL in were found in histology. All the EAC and AIS were either HPV16 or HPV 18 positive. No high-grade histological lesions were found among the hrHPV-negative patients diagnosed with cytological endocervical cell atypia. A later database search revealed one HPV-negative gastric-type mucinous adenocarcinoma that was missed by the primary HPV screening.


D. vulgaris str. Hildenborough. The results of this study improved the current understanding of dynamic interactions between prophages and CRISPR with their hosts in both cultured and hitherto-uncultured SRP strains, and how their distribution affects the microbial community dynamics in several sulfidogenic natural and engineered environments.Sulfate reducing prokaryotes (SRP) are a phylogenetically and physiologically diverse group of microorganisms that use sulfate as an electron acceptor. SRP have long been recognized as key players of the carbon and sulfur cycles, and more recently, they have been identified to play a relevant role as part of syntrophic and symbiotic relations and the human microbiome. Despite their environmental relevance, there is a poor understanding about the prevalence of prophages and CRISPR arrays and how their distribution and dynamic affect the ecological role of SRP. We addressed this question by analyzing the results of a comprehensive survey of prophages and CRISPR in a total of 91 genomes of SRP with several genotypic, phenotypic, and physiological traits, including genome size, cell volume, minimum doubling time, cell wall, and habitat, among others. Our analysis discovered 81 prophages in 51 strains, representing the 56% of the total evaluated strains. Prophages are non-uniformly distributed across the SRP phylogeny, where prophage-rich lineages belonged to Desulfovibrionaceae and Peptococcaceae. Furthermore, our study found 160 CRISPR arrays in 71 SRP, which is more abundant and widely spread than previously expected. Although there is no correlation between presence and abundance of prophages and CRISPR arrays at the strain level, our analysis showed that there is a directly proportional relation between cellular volumes and number of prophages per cell. This result suggests that there is an additional selective pressure for strains with smaller cells to get rid of foreign DNA, such as prophages, but not CRISPR, due to less availability of cellular resources. Analysis of the prophage genes encoding viral structural proteins reported that 44% of SRP prophages are classified as Myoviridae, and comparative analysis showed high level of homology, but not synteny, among prophages belonging to the Family Desulfovibrionaceae. We further recovered viral-like particles and structures that resemble outer membrane vesicles from  For many years, bacteriophages have been considered the dark matter of the biological world due to their high abundance, ubiquitousness, dynamic population, genetic diversity, and indecipherable mosaic genomic architecture ,2. The tThe prophage–host interaction can have positive or negative effects for both the host and the virus, which depends on the environment. On one hand, carrying integrated phages that kill the lysogenic cell after eventual induction is considered an extreme relationship for the host, but it is convenient way to silently replicate inside a protective cellular environment for the virus. Moreover, replication and maintenance of extra bacterial DNA is a metabolic burden that affects the long-term fitness of the host. On the other hand, carrying prophages can result on several advantages. For example, it has been shown that prophages can protect host against further viral infections, and phagocytosis ,6,8, as Sulfate reducing prokaryotes (SRP) are a phylogenetically, physiologically, and nutritionally diverse group of microorganisms that have in common the use of sulfate as an electron acceptor, which results in the production of sulfide as the end-product ,14. The Desulfovibrio vulgaris str. Hildenborough 1/p), where p is equal to 1.6075. SRP were classified as small surface area (<6 μm3), middle surface area (6–12 μm3), and large surface area (>12 μm3).An extensive meta-analysis of morphological, physiological and metabolic features and lifestyles of the SRP was performed based on the information for the 91 individual species derived from literature. This analysis included growth conditions, such as pH ranges and temperature ranges ; cell morphology aspects, such as Gram staining properties of the cell wall (“Gram-positive” or “Gram-negative”), motility (presence or absence of flagella), cell shape ; metabolic traits, such as electron acceptors and electron donors for growth (with the terms “complete” or “incomplete” oxidizer); and growth characteristics, such as the doubling time for optimum growth that was estimated from research that included culturing of pure strains. According with those estimations, SRP were grouped in two categories based on the capability to duplicate (under optimum conditions) in ≤12 h or >12 h, as fast-growing or slow-growing strains, respectively. In addition, the analysis included the source of isolation for each strain, or the habitat where material that allowed the reconstruction of the genomes was obtained, as in the case of Candidatus t-test for difference of means, and Wilcoxon test. PCA was used to gain some understanding of how ecophysiological traits may affect lysogeny and the prevalence of CRISPR elements in the SRP community. PCA is a technique used in dimension reduction when there are many variables. Commonly, a few dimensions can explain 80% or more of the variability of the data. In addition, the graphical visualization of the first two components provides a suitable tool to detect hidden/unknown patterns among several variables [t-test and Wilcoxon tests, and parametric and non-parametric techniques, respectively, are tools to assess the difference between two variables. In these tests, the null hypothesis is that the centers of both variables are equal. For the t-test, the difference between two variables is measured through the difference in averages of each variable, which is standardized by the standard deviation, while the Wilcoxon test is based on the location-shift, meaning that the distribution of the two variables is the same except for a constant. Both tests have advantages and disadvantages, as well as their assumptions, and therefore, their use should be adjusted appropriately to each aim. The t-test assumes independence between each observation and the normality of both variables. The Wilcoxon test assumes independence between each observation and identical distribution in both populations, but not necessarily normal distribution. The principal advantage of the t-test, over the Wilcoxon test, is their higher power statistic (reject when you have to reject) but is very poorly robust in the presence of outliers. The principal advantage of the Wilcoxon test is its robustness in presence of outliers [The statistical techniques used in this paper are Principal Component Analysis (PCA), ariables ,47. The outliers ,49,50,51outliers .Desulfovibrio vulgaris str. Hildenborough (DSM 644), Desulfocurvibacter africanus 2603 (DSM 2603), Desulfovibrio desufuricans subsp. aestuarii (DSM 10141) were obtained from the DSMZ, and cultured at 30 °C under strict anaerobic conditions, as previously described [D. vulgaris str. Hildenborough and Desulfocurvibacter africanus 2603 were cultured in LS4D medium [Desulfovibrio desulfuricans subsp. aestuarii was cultured in 195-C medium with strain specific modifications. We evaluated the possible induction of phages using increasing amounts of mitomycin added in a final concentration of 1 to 5 micrograms mL−1 ). The other six could not be taxonomically assigned. Furthermore, 75% of those small prophages were present in strains carrying a single prophage, therefore, probably a high fraction of those small prophages are either mobile genetic elements or prophage remnants that may represent ancient insertion events that have lost their ability to induce [In order to evaluate the prevalence of prophage elements in SRPs, in this study a comprehensive survey in 91 publicly available SRP genomes was conducted. The analysis using PhiSpy and PHASTER softwares resulted in the discovery of 81 prophage elements in 51 SRP strains, representing the 56% of the evaluated strains . Thirty-ectively . Twelve Desulfotomaculum acetoxidans DSM 771 and Desulfotomaculum reducens MI-1 that possess more than three prophages. In addition to the high prevalence, prophages that reside in Peptococcaceae and Desulfobulbaceae strains contain significantly larger prophages in comparison to the other SRP families .Prophages are non-uniformly distributed across the SRP phylogeny, where few SRP clusters concentrate a high number of prophages . While pDesulfovibrio strains are grouped in two clusters according to the phylogeny. Cluster number one included two groups of Desulfovibrio strains. The first group includes a stable monophylethic set of 13 strains together with five Desulfovibrio strains  that branched more independently but have been isolated from more similar environments of strains belonging to cluster 1 than the environment of strains that belong to the cluster 2. In cluster 1, prophages are present in 13 out of 18 strains covering a total of 29 prophages , Walvis Bay (South Africa), and Venice , respectively, D. acetoxidans DSM 11,109 was isolated from a UASB sludge reactor [D. salexigens, D. bastinii DSM 16055, D. putealis DSM 16056, Desulfocurvus vexinensis DSM 17965, Desulfomicrobium baculatum DSM 4028, Desulfovibrio sp. X2, Desulfurivibrio alkaliphilus AHT 2, and Desulfococcus oleovorans Hxd3. Most of these SRP are incomplete oxidizers, slow growers and have flagella . This cluster comprises lysogens belonging to the genus Desulfotomaculum (Family Peptococcaceae), including the poly-lysogenic D. acetoxidans DSM 771, D. hydrothermale Lam5, D. reducens MI-1, and D. ruminis DSM 2154, which have additional 11 prophages (Desulfobulbus propionicus type strain (1pr3), all other strains carry only one prophage per genome. Members of cluster 3 have a broader range of ecophysiological traits compared with clusters 1 and 2. The cluster includes two thermophiles, Thermodesulfobacterium commune DSM 2178, and Desulfotomaculum hydrothermale Lam5, both incomplete oxidizers isolated from volcanic material from Yellowstone and a hot spring in North-east Tunisia, respectively [D. reducens MI-1 (3) and D. hydrothermale Lam5 (2) have cell wall structure typical of the Gram-positive bacteria [The cluster number 3 is restricted to strains belonging to Thermodesulfobacteria, Syntrophobactaraceae, Desulfobulbaceae that belong to the rophages . Clusterectively ,63. Lytiectively . Furtherbacteria . ClusterDesulfobulbus propionicus type strain (1pr3) that possess flexible degradative machinery to metabolize different substrates. Indeed, D. propionicus type strain (1pr3) is capable to oxidize propionate to acetate without dependence of syntrophic interactions [Syntrophobacter fumaroxidans MPOB that is capable to grow on propionate axenically or syntrophically with other microorganisms, in a process that involves the efficient scavenging of products to cope with the thermodynamic constraint of the propionate oxidation to acetate [S. fumaroxidans MPOB partners with methanogens, such as Methanospirillum hungatei, Methanobacterium formicicum, or with other sulfate reducing bacteria such as Desulfovibrio desulfuricans strain G11. In these syntrophic associations, S. fumaroxidans MPOB plays a role of the hydrogen or formate scavenger to keep them at a concentration that makes propionate degradation to be energetically feasible [S. fumaroxidans MPOB (prophage 83R1) against the genome of D. desulfuricans strain G11 using tBLASTn showed that 17 out of the 55 ORFs predicted for the prophage are homologs of genes from D. desulfuricans strain G11, including genes encoding for the minor capsid protein, major head protein, endonuclease, and phage protease (Data Set 6). It could be interesting to couple the metabolic characterization with in-depth ecological characterization of syntrophic partnerships, which could open opportunities to gain insights into the occurrence and distribution of prophages shared between syntrophs, and how those mechanisms may help them to improve the nutritional interplay in the environment. These observations suggest that, in addition to the broad metabolic flexibility present in lysogenic SRP of cluster 1 and 2, the lysogens of cluster 3 possess physiological flexibility, allowing them to thrive under a wide range of temperatures, salt concentrations, pH, life-styles, and also cope with a broad range of toxic compounds.Two lysogens belonging to cluster 3 are capable of degrading propionate. One of those strains is ractions , and alsractions . The sec acetate ,69,70. SClustered regularly interspaced short palindromic repeats (CRISPR) is a sophisticated adaptive immune defense system that allow the identification and cleavage of allochthonous DNA, storing the sequences of the invader in series of spacers separated by repeats that permits the host to quickly identify and react upon re-exposure . In contKlebsiella species contain CRISPR-Cas systems [D. acetoxidans DSM 771 and D. ruminis DSM 2154, were found to contain the higher quantity of CRISPR arrays with a total of 11 and 10, respectively  of those species lack CRISPR arrangements, suggesting that other mechanisms may be reinforce for defense against virus in SRP belonging to Desulfovibrionaceae , the distribution of the total number of prophages and CRISPR arrays followed a similar order at the level of Family   . Meanwhi 0.0031) , for whiYersinia pestis and Vibrio vulnificus [cas genes were previously reported as present in the megaplasmid of D. vulgaris Hildeborough, but a later report as well as our findings did not detect any CRISPR array in the plasmid [In agreement to prior studies based on a wider set of data , our datlnificus . Indeed, plasmid ,76. FurtSo far, our results suggested that the trade-off between the energetic burden of maintaining prophages and CRISPR loci versus their contribution to the fitness of the host may be driven by a balance of physiological and environmental factors. Furthermore, we hypothesized that several ecophysiological traits are important to shape the distribution of prophages and CRISPR in SRP. This hypothesis was addressed applying a species-specific approach, retrieving and analyzing several phenotypical and physiological traits from both environmental and experimental studies of the 91 SRP included in the study. These data include type of cell wall (based on Gram stain), motility (presence of flagella), optimum pH and T° for growth, minimum doubling time, cell volume, surface cell area, morphology type, and habitat, based on the environment where strains were either found or isolated .Chlamydomonas eustigma, an acidophilic green alga, in contrast to evolutionarily related neutrophilic green algae [Desulfotomaculum) are found in the upper size of the PCA, in contrast to a group of copiotrophic strains that are found in the bottom  .Principal Component Analysis (PCA) of the eight variables of lysogenic SRP strains showed the first component (PC1) accounted for 25.8% of the total variation in the dataset, and it is highly influenced by genome size, temperature of growth, and cellular volume. Therefore, lysogenic SRP located in the left side contain bigger genomic size and cell volume and often reside in mesophilic environments, in contrast to those lysogenic SRP of the right side of the PCA . The secen algae . Conseque bottom . Interes0.03435) , indicat0.03435) . This fi3) compared to strains of other families (Median size ≤ 1.68 μm3), whereas the median of genome size of all the strains of SRPs belonging to the Desulfobacteraceae, Desulfohalobiaceae, Desulfovibrionaceae, Desulfobulbaceae, and Peptococcaceae families ranged between 3,916,410 and 3,969,010 bp . While motility and growth rate are not relevant  of prevalence in mesophiles  among the SRP community. These results are in agreement with previous studies [D. vulgaris Hildenborough were classified in the order Caudovirales. While prophages 1R1, 1R2, 1R5, and 1R6 were classified as Myoviridae, 1R3 and 1R4 were classified as Siphoviridae and Podoviridae, respectively. In addition to D. vulgaris str. Hildenborough phages, other SRP phages have been previously isolated. This is the case of like-phages particles induced from pure cultures of D. desulfuricans subsp. desulfuricans str. ATCC 27,774 [Desulfovibrio alaskensis G20 [Desulfovibrio salexigens, a salt-requiring SRP [D. aespoeensis Aspo-2, which was isolated from the same environment [This study indicates that the model strain  studies . The disC 27,774 , and mornsis G20 . Kazimurring SRP . Furtherironment . Howeverp-value = 0.0014 of Wilcoxon test), suggesting that integrases, as well as other essential phage genes, may have been lost after integration. In addition, many proteins associated with the viral structural proteins are present in the prophages, including head-associated (66), capsid-associated (44), tail-associated (266), and baseplate-associated (69) proteins. SRP prophages contain many genes that were annotated as hypothetical proteins or unknown functions, in agreement with previous studies. For example, the six prophages of D. vulgaris Hildenborough averaged 45% of the genes classified as hypothetical proteins or conserved hypothetical proteins, while the three prophages of D. vulgaris DP4 averaged 41% genes encoding hypothetical proteins. However, there are some prophages with higher percentage of genes with unknown function. This is the case of the prophages in D. africanus DSM 2603 and D. vulgaris Miyazaki, for which the genes with unknown functions covered a total of 57% and 54% of the genome, respectively , a substantial set of proteins is unique across the prophage SRP community  added at early exponential phase (D. vulgaris str. Hildenborough. In contrast, the presence of the antibiotic did not affect the growth of D. africanus 2603 and D. acrylicus DSM 10141. Viral-like particles with similar morphotype of Podoviridae and structures that resembles outer membrane vesicles were observed in TEM micrographs of D. vulgaris str. Hildenborough after induction with mitomycin C (D. alaskensis DSM16109, in cultures incubated in presence or absence of mitomycin C [D. vulgaris str. Hildenborough and D. africanus 2603 slightly reduced the growth rate of D. africanus 2603, only when the extract was added during exponential phase (In this study, numerous assays to induce phages from SRP cultures were carried out. These attempts included induction of virus using mitomycin C in three strains of the al phase –C. The eomycin C . Membranomycin C . A previomycin C . Afterwaal phase . Double-Desulfovibrio piger ATCC 29,098 or D. desulfuricans subsp. aestuarii ATCC 29578, respectively [From an ecological perspective, the composition and dynamic of naturally occurring communities in pristine sulfidogenic environments are mainly shaped by two forces. The bottom up force selects for SRP that are either specialized for a limited number of substrates or capable to utilize a wide variety of nutrients, such as ectively ,97. Thusectively ,90,98. Sectively , especiaectively . From th


The study is designed to understand the situation of full-term infants breastfeeding within 6 months of birth in Xi'an before the Covid-19 pandemic and analyze the influencing factors of exclusive breastfeeding.Five hospitals in Xi'an province have been selected as research centers. Full-term infants who met the inclusion and exclusion criteria were recruited from these centers between January 1 and February 28, 2019. The feeding situation at 10 days, 42 days, 3 months, and 6 months after birth were investigated. A self-designed breastfeeding questionnaire was used for investigation and follow-up. SPSS 22.0 was applied for statistical analysis of the data.The exclusive breastfeeding rate of full-term infants on days 10 and 42 and at months three and six after birth was 61.38%, 54.78%, 48.83%, and 38.78%, respectively, with a decreasing trend over time. During breastfeeding within 48 h after delivery, 1,653 cases (91.83%) of puerpera had different grades of pain, including 1,325 cases (80.16%) of mild discomfort, 321 cases (19.42%) of moderate pain, and seven cases (0.42%) of severe pain. Within 24–48 h postpartum, 1,607 (89.27%) mothers faced problems related to postpartum breastfeeding. Among them, 694  neonates could not be fed effectively; 665 (41.38%) mothers had wound pain and had inconvenience to turn over; 598 (37.21%) neonates were difficult to wake up; 439 (27.32%) mothers had incorrect feeding posture; 181 (11.26%) mothers experienced other problems. The Cox risk regression model showed that weight gain during pregnancy was higher than the recommended standard. Living in suburban counties was a risk factor of exclusive breastfeeding for full-term infants. Participation in breastfeeding courses during pregnancy, feeding more than eight times daily after delivery, were the protective factors of exclusive breastfeeding for full-term infants.The body weight gain of parturients should be controlled within a reasonable range during pregnancy. Parturients were encouraged by medical staff to participate in breastfeeding courses or watch the breastfeeding process during pregnancy to increase their self-confidence and improve the rate of exclusive breastfeeding for full-term infants. In addition, it is necessary to strengthen the publicity of breastfeeding in suburban areas to promote breastfeeding. The death of about two-thirds of children <5 years of age are caused by infections, and about 35% are related to malnutrition worldwide . In contThe rate of EBF in China within 6 months varies among different regions. Although it shows an increasing trend, rate is still lower than 50%. In 2010, a survey of 2,354 babies in 12 provinces (including Shanxi) in the central and western regions of China showed that only 28.7% of the infants received EBF within 6 months postpartum . In 2012Therefore, this study was designed to investigate the current breastfeeding situation of full-term infants within 6 months in Xi'an and analyze its influencing factors to provide a basis for establishing breastfeeding measures and improving infant feeding in clinical practice.A stratified sampling method was used to collect pregnant women and newborns after delivery during the third trimester of the study period. According to previous literature, we assumed that the EBF rate is 30%, and the formula for estimating the overall rate is as follows: The total number of subjects required for investigation (considering 20% loss-to-follow-up rate) was 1,609. According to the number of delivery cases of the hospitals in 2018, one provincial-level maternal and child health hospital, three municipal hospitals, and one county-level maternal and child health hospital were selected. The sample size of each hospital was calculated based on the proportion of the number of delivery cases of this hospital in all selected ones.The full-term infants born from January 1, 2019, to February 28, 2019, and their mothers were selected as subjects at five hospitals in Xi'an, including one provincial hospital, three municipal hospitals, and one county hospital. All 5 hospitals are baby-friendly hospitals. The outpatient health education includes training sessions for pregnant women, such as the structure of breasts in different periods, breastfeeding, the process of natural childbirth, etc. After being admitted to the hospital, pregnant women were instructed with breastfeeding knowledge by the nurses. The Early Essential Newborn Care is implemented immediately after the baby is born in the delivery room. In 2 hospitals , babies were placed on the mothers' abdomen after born and the skin contact time was no <90 min after the baby was born. In 3 hospitals , the skin contact time was no <30 min. The nurses then wiped and weighed the newborns. After returning to the ward, mothers were instructed with breastfeeding practice guidance by nurses in a “one-on-one” mode. In cases with cesarean section, mothers returned to the ward, and skin contact and sucking were immediately carried out. All hospitals implemented 24-h rooming with mothers and babies. Breastfeeding consultations were provided through follow-up visits and telephone calls for 1 week after discharge.A self-designed breastfeeding questionnaire and follow-up questionnaire were used for investigation. Eighteen hundred questionnaires with complete information were collected, all of which were continuous cases.Inclusion criteria:① Single-fetus full-term infants with a gestational age of at least 37 weeks; ② their parents were married and voluntarily participated in the breastfeeding investigation and were willing to cooperate with the investigators.Exclusion criteria:① Twins, or singles with a gestational age <37 weeks; ② their parents were unwilling to participate in the investigation; ③ mothers with infectious diseases such as HBV, HCV, etc.; ④ pregnant women participating in other studies.This study was an observational study, in which self-designed breastfeeding questionnaire and follow-up questionnaire were used. This study was approved by the local ethic committee and all subjects provided written informed consent before enrollment. The breastfeeding questionnaire included the demographic characteristics of pregnant women and their husbands , general conditions of pregnant women during pregnancy , postpartum lactation-related surveys , and general information of babies. Before the study, a one-month pre-survey was performed in the provincial hospital from November to December 2018, and four rounds of revision of the questionnaire were completed by the chief nurses and investigators of the ward who participated in this study. The final version of the revised questionnaire was then used. The questionnaire was divided into two parts. The first part was an investigation of general information using the Breastfeeding Questionnaire, which included demographic characteristics , pregnancy , and breastfeeding-related investigations. The second part was the follow-up investigation of newborns from birth to 6 months.A total of 2,520 full-term infants were included in this study and 1,800 cases were followed for 6 months . The queThe pilot investigation was conducted in this study, and the questionnaire was completed at the Northwest Women's and Children's Hospital in December 2018. All team investigators received unified training, including preparations before the investigation, investigation precautions, review of questionnaires, handling of special situations, investigation methods, etc. In the on-site investigation process, the investigator maintained an objective attitude to avoid leading questions and inquired item by item according to all originally designed questionnaires, and timely checked the questionnaire. The missing and wrong items were corrected immediately once found. A double data entry method was adopted to enter the database.All definitions of the terms are listed in The feeding patterns of infants aged 1–6 months were divided into EBF group , partial breastfeeding group  and artificial feeding group (not Including breast milk during feeding).2 indicates underweight, 18.5–23.9 kg/m2 indicates normal weight, 24.0–27.9 kg/m2 indicates overweight, and ≥ 28 kg/m2 indicates obesity , normal GWG, and excessive GWG , which assessed the pain based on the subject's own perspective. The total score of the scale is 0–10 points: 1–3, mild pain; 4–6, moderate pain; 7–10, severe pain.EBF means that the baby should not have other food or drinks except breastmilk and is allowed to take vitamins, minerals, and medicine, except for water intake.The EBF rate = the number of infants in the same period, still receiving EBF 24 h before the investigation / number of infants in the same period ×100%.The feeding patterns of infants aged from 1 to 6 months in this study were divided into the EBF group , the partial breastfeeding group , and the artificial feeding group .Pregnancy complications included gestational diabetes, pregnancy hypertension, hypothyroidism, hyperthyroidism, placenta previa, and intrahepatic cholestasis.During pregnancy, attending a breastfeeding course refers to watching a breastfeeding promotion video, a breastfeeding course video, or witnessing a friend breastfeeding an infant.Nipple retraction: in retraction Grade I, part of the nipple is inverted, with an existing nipple neck that can be easily squeezed out. The size of the nipple after being squeezed out is average. In retraction Grade II, the nipple is completely inverted in the areola, but the nipple can be squeezed out by hand. The nipple is smaller than average, most without a nipple neck. In retraction Grade III, the nipple is completely buried in the areola, making the inverted nipple unable to be squeezed out .2].Pre-pregnancy BMI was calculated as follows: pre-pregnancy BMI = pre-pregnancy weight (kg)/[height (m)2 is considered underweight, 18.6–23.9 kg/m2 is considered average, 24.0–27.9 kg/m2 is considered overweight, and ≥28 kg/m2 is considered obese  – pre-pregnancy weight (kg).According to the 2009 Institute of Medicine guidelines, the recommended weight gain standards for underweight, average weight, overweight, and obese women were 12.5–18.0 kg, 11.5–16.0 kg, 7.0–11.5 kg, and 5.0–9.0 kg, respectively. The weight lower than, in line with, and higher than the above-recommended standards was considered insufficient GWG, normal GWG, and excessive GWG, respectively (The postpartum breastfeeding pain score in 48 h: mild pain (1–3), moderate pain (4–6), and severe pain (7–10).EpiData 3.0 was used to establish the database by double data entry. The classified data were expressed as the rate, and the continuous data were expressed as mean ± standard deviation. Univariate analysis was performed using the survival analysis K–M method, and multivariate analysis was performed using the Cox survival risk model. Before applying the Cox proportional hazard model, the time-dependent model was used to verify the proportional risk of the above factors, which all met the equal proportional risk hypothesis. Therefore, the Cox regression was included. All statistical analyses were performed using SPSS 22.0 software. For bilateral inspection, the inspection level was set as α = 0.05.2, and in the third trimester was 26.71 ± 3.43 kg/m2, with an average weight increase of 13.63 ± 5.40 kg. The mothers and their husbands' occupations' were mostly company employees, accounting for 34.05% and 40.00%, respectively. There were 758 people with maternity leave ≥150 days, accounting for 42.11%. Complications occurred in 678 people (37.67%). The four primary complications were 275 (15.27%) with gestational diabetes, 142 (7.89%) with hypothyroidism, 85 (4.72%) with placenta previa, and 49 (2.72%) with hypertension during pregnancy. See This study was a descriptive study in a non-experimental manner. The distribution of puerpera in this study was 925 cases (51.38%) at provincial hospitals, 645 patients (35.83%) at municipal hospitals, 230 cases (12.78%) at county health hospitals. The mean pre-pregnancy BMI was 21.50 ± 3.08 kg/mAn investigation was made on the present condition of postpartum mothers' breasts, initiation of lactation, lactation pain, and cracked nipples. The results showed that 21.72% of the parturients had initiation of postpartum lactation stage II. A total of 13.22% of parturients suffered from nipple depression, and Grade I retraction was dominant, accounting for 67.64%. A total of 91.83% of parturients had pain during lactation. There were 281 cases (15.61%) of cracked nipples due to neonatal sucking. Within 24 to 48 h postpartum, 1,607 (89.27%) mothers faced problems related to postpartum breastfeeding. Among them, 694  neonates could not be fed effectively; 665 (41.38%) mothers had wound pain and had inconvenience to turn over; 598 (37.21%) neonates were difficult to wake up; 439 (27.32%) mothers had incorrect feeding posture; 181 (11.26%) mothers experienced other problems see .We further investigated the reasons why infants were fed with formula powder during follow-up. At 10 days, 42 days, 3 months, and 6 months postpartum, the follow-up data were recorded. As shown in Postpartum follow-up showed that the rate of EBF decreased from 61.39% to 38.78% over time, showing a decreasing trend, as shown in Whether the infant was exclusively breastfed within 6 months was taken as the outcome variable Y . The univariate survival analysis (K–M method) was performed using 16 factors, including age, pre-pregnancy BMI, pregnancy weight gain, parturients' education, husbands' education, family monthly income, residence, whether primiparae, the mode of delivery, complications during pregnancy, feeding suggestions for husband, feeding suggestions for family members, participation in breastfeeding courses during pregnancy, the frequency of daily breastfeeding 48 h after delivery, the planned breastfeeding mode 6 months after delivery, and the scheduled breastfeeding time . The resWhether the infant was exclusively breastfed within 6 months was taken as the outcome variable Y . The above 16 factors were subjected to multivariate Cox regression, with the assigned values shown in A cohort study in northwest China from 2007 to 2010 showed that the EBF rate of newborns after 14 days was 24%, and after six months, only 3% . A summaThe systematic evaluation by Cohen et al. showed that the influencing factors for the initiation and continuation of breastfeeding mainly included smoking, mode of delivery, multiple births, parturients' education, and breastfeeding education . This stThe results of this study showed that when the parturients' weight gains during pregnancy with different BMI ranges before pregnancy were higher than the recommended standards, the risk of non-EBF within 6 months after delivery was 1.186 times higher than those with weight gains meeting the recommended standards. Huang  found thP = 0.00), with a statistically significant difference. Due to the imbalance in economic and educational development between cities and suburban counties, the difference in the acceptance and execution of the concept of feeding among the population, and the publicity of various milk powder advertisements, the belief in EBF is easily shaken, thus reducing the rate of EBF in suburban counties. Therefore, it is necessary to take appropriate intervention measures to improve the knowledge and practices of parturients and family members on breastfeeding in suburban areas to increase the rate of EBF and prolong breastfeeding time.Since the “two-child policy,” the state has advocated breastfeeding to reduce infant mortality and improve infant health conditions. The latest study shows that the infant mortality rate (<1 years-old/thousand) in rural areas is more than twice that in urban areas . Other sThis study mainly investigated the EBF rate of full-term infants within 6 months in the Xi'an area and analyzed its influencing factors to provide the basis for the clinical designation of targeted measures. However, during the research, due to the adjustment of obstetrics work mode in some hospitals, the final collected data was less than the planned cases. After the unqualified samples were excluded, the qualified samples were fewer. A total of 326 parturients were lost within the 6-month follow-up period with a rate of 12.9%. Most parturients were lost to follow-up in the sixth month, which might be related to the decrease of parturients' cooperation with a prolonged study. The above two points may limit the outcome of this study.Our work fills the gap in the rate of exclusive breastfeeding for full-term infants in Xi'an, China. This study showed that the EBF rates in Xi'an at 10 days, 42 days, 3 months, and 6 months after birth were 61.39%, 54.78%, 48.83%, and 38.78%, respectively. There is a downward trend over time. Although the EBF rate within 6 months has increased compared with previous reported data, there is still a gap with the WHO's target of 50% within 6 months. Our study also found that weight gain during pregnancy higher than the recommended standard, and living in a suburban county was a risk factor for mothers to terminate exclusive breastfeeding within 6 months after delivery, while taking breastfeeding courses during pregnancy, and the number of breastfeeds per day within 48 hours after delivery was >8 were the factors contributing to continuous EBF for 6 months postpartum. With the changes in people's eating habits and dietary structure, excessive intake of fats and oils during pregnancy may lead to excessive weight gain and excessive weight gain, and may cause early termination of EBF after delivery. Wang et al. investigated postpartum lactation in 240 parturients and found that high BMI and high GWG before pregnancy are risk factors for delayed lactation initiation. Primiparas were more prone to delayed onset of lactogenesis . It has The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethics Committee of Northwest Women's and Children's Hospital (20-078). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.LM, W-LG, and Z-WY: conception and design of the research. X-YL, X-QZ, J-JZ, and JY: acquisition of data. Z-WY: analysis and interpretation of the data and writing of the manuscript. Z-WY and P-FQ: statistical analysis. LM: obtaining financing and critical revision of the manuscript for intellectual content. All authors read and approved the final draft.This study was funded by Shaanxi Province Key Research and Development Program General Project–Social Development (2018SF-224). The funding body had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Murrah breed of buffalo is an excellent dairy germplasm known for its superior milk quality in terms of milk fat and solids-not-fat (SNF); however, it is often reported that Indian buffaloes had lower lactation and fertility potential compared to the non-native cattle of the country. Recent techniques, particularly the genome-wide association studies (GWAS), to identify genomic variations associated with lactation and fertility traits offer prospects for systematic improvement of buffalo. DNA samples were sequenced using the double-digestion restriction-associated DNA (RAD) tag genotyping-by-sequencing. The bioinformatics pipeline was standardized to call the variants, and single-nucleotide polymorphisms (SNPs) qualifying the stringent quality check measures were retained for GWAS. Over 38,000 SNPs were used to perform GWAS on the first two principal components of test-day records of milk yields, fat percentages, and SNF percentages, separately. GWAS was also performed on 305 days’ milk yield; lactation persistency was estimated through the rate of decline after attaining the peak yield method, along with three other standard methods; and breeding efficiency, post-partum breeding interval, and age at sexual maturity were considered fertility traits. Significant association of SNPs was observed for the first principal component, explaining the maximum proportion of variation in milk yield. Furthermore, some potential genomic regions were identified to have a potential role in regulating milk yield and fertility in Murrah. Identification of such genomic regions shall help in carrying out an early selection of high-yielding persistent Murrah buffaloes and, in the long run, would be helpful in shaping their future genetic improvement programs. Bubalus bubalis) is an imperative livestock species and act as a key component for improving agricultural economy and supplying milk, meat, and draft power. The buffalo population across the world was recently estimated to be 194 million, 97% of which were present in Asia  in buffaloes for lactation traits have been mainly limited to the use of bovine single-nucleotide polymorphism (SNP) chip  and AffyHence, the present study was conducted to identify novel SNPs associated with milk production, composition, lactation persistency, and fertility traits at the genomic level using the genotype-by-sequencing technique in Murrah buffalo, India’s major buffalo breed and milch animal of the nation.A total of 672 test-day records on each trait, i.e., milk yield (TDMY), fat percentage (TDFP), and solids-not-fat (SNF) percentage (TDSNF) were collected from 96 female Murrah buffaloes reared at LRC, NDRI, Karnal, India (29.68°N and 76.99°E). Records of 96 buffaloes on 305 days’ milk yield (305DMY), birth weight (bwt), age at first calving (AFC), calving interval (CI), and age at sexual maturity (ASM) in months were collected. Other traits such as lactation persistency, postpartum breeding interval in days (PPBI), and breeding efficiency (BE) were derived from the primary phenotype records. Breeding efficiency was calculated for female buffaloes as described by Lactation persistency was estimated by following four different methods:(I)(II)(III)(IV)SphI and MluCI restriction enzymes. Adapters (P1 and P2) were prepared as per standard Illumina read multiplexing protocol using an inline barcode along with Illumina index for library preparation. After adapter ligation and size selection, samples were sequenced on a Illumina Hi-Seq 2000 platform.DNA was isolated from 96 Murrah buffaloes selected for the study following the standard phenol-chloroform method . SamplesGCF_003121395.1, was retrieved from the NCBI dataset and used as a reference genome. Index and sequence dictionary files were created using the Burrows–Wheeler algorithm (BWA)   and Picag FastQC , and eacg FastQC . Adapterg FastQC .3 The BWqualimap . Variantqualimap .p < 0.0001 were removed from the dataset. SNPs in LD with r2 > 0.8 were also removed. Only autosomal and X chromosome SNPs were retained for the final analysis. All the quality control operations and data preprocessing were performed using PLINK v1.9  of TDMYs, TDFPs, TDSNFs, and 305DMY; lactation persistency calculated by four different methods; age at sexual maturity in months; postpartum breeding interval (in days); and breeding efficiency. Genome-wide identity-by-state (IBS) for all pairs of individuals was checked. Multidimensional scaling (MDS) based on SNP information was done to check for the presence of any population stratification  at 5 and 10% levels ( ≤ (i/m) × q, where i = rank of the SNPs, m = no. of individual tests performed, and q = either 0.05 or 0.1  instead of direct traits. Principal component analysis (PCA) was performed in the R programming environment (v4.0.3) on the 672 test-day records of each trait separately. As the first two PCs cumulatively account for most of the variation in the dataset, they were selected to be GWAS traits. The traits on which GWAS were performed were PCs . After quality check of raw data, it was observed that the average GC content was 50.54%. The rate of duplication was quite high, i.e., 80.11%, which was later checked during variant calling. The quality score (Q) throughout the dataset varied from 35 to 40.Raw files that were quality checked in FastQC were combined using MultiQC, and the report is provided as 1 and PC2) of TDMY, TDFP, and TDSNF.GWAS were performed with 38,560 SNPs, and the genome-wide significant threshold was set at the 5% FDR level. GWAS were performed on the two foremost variation explaining PCs , 9 (62699319 bp), and 16 (74362861 bp) were significantly associated with PC1 and PC2 of the test day’s milk yield were also in the top SNP list for 305DMY. The Manhattan and Q-Q plots for association results of 305DMY are given in GWAS on 305DMY revealed that there was no significant association between SNPs and the trait. It was observed that five SNPs that appeared in the list with GWAS for PCp-values along with their genomic position and genes within a ± 20-kb region are listed in GWAS on lactation persistency estimated by four different methods revealed no significant association between SNPs and the trait generated. The top 10 SNPs having the lowest p-values and genes within a ± 20-kb region are listed in Upon performing GWAS on age at sexual maturity, postpartum breeding interval, and breeding efficiency, no significant association of any SNPs were observed for the traits; however, the top 10 SNPs with the lowest The test-day model (TDM) as repeated measurements of milk yield traits is the method of choice for predicting 305 days’ milk yield. TDMs account for environmental effects on each test day and is useful to model individual lactation curves . PCA beiGRIA3, was reported by It is important to understand the reasons for considering 96 samples taken in the study as optimum, as in the buffalo farming scenario in Asia, particularly in India, the maximum number of buffaloes maintained at any large organized herd ranges from 250 to 500, with 100–200 breedable buffaloes having complete phenotype information. The National Dairy Research Institute has the second-largest herd in the Indian Council of Agricultural Research (ICAR) with a well-managed herd of 250 breedable buffaloes. Furthermore, there is no buffalo sequencing consortium/project in operation (India). In such a case, one could afford this sample size to genotype with complete pedigree and with sufficient genetic diversity. However, the sample size is less for conducting GWAS and raises the question of whether the results are reliable enough. It was observed that the results obtained under the present study are encouraging and important, as one of the genes identified in the present study, i.e., To identify novel SNPs associated with various economic traits, sequencing through the ddRAD approach was performed. LOC100847171, BCL6, RTP2, SST, PTGS2, LOC100295047, and LOC101908004 are associated with milk production; KCTD8, LOC782855, LOC101904777, ESRRG, TRNAY-AUA, and GPATCH2 are associated with fat percentage; LOC101903483, SART3, ISCU, CMKLR1, WSCD2, MFNG, CARD10, and USP18 are associated with protein percentage. MFSD14A, SLC35A3, PALMD, RGS22, and VPS13B, for milk production in the Italian Mediterranean buffalo through GWAS. In another GWAS in buffalo, D-glucose level in the blood affect milk production in buffalo.An earlier bovine SNP chip was used to study the traits of buffalo. Using a bovine SNP chip (Illumina BovineSNP50 BeadChip) for a GWAS in buffalo population, 1 of TDMY at the 5% FDR level. However, six SNPs present on chromosomes 1, 6, and 9 at 182,059,836, 35,648,660, and 48,559,942 bp, respectively, were above the suggestive threshold of 10% FDR and are presented in 1 of TDMY. However, for the rest of the traits studied, no SNPs were detected to be significantly associated (or as rejections) at 5% or even 10% FDR.In the present study, GWAS performed on the milk yield PCs explaining maximum variation, which revealed that three SNPs were significantly associated with PCGRIA3 gene was found. Glutamate ionotropic receptor AMPA type subunit 3 (GRIA3) is reported to be very significantly associated with daughter pregnancy rate in US Holstein cows  glutamate receptors, thus regulating trafficking of AMPA receptors. AMPA receptors are known to participate in luteinizing hormone (LH) secretion  pathways involved in mammary gland development. In the present study, lactation persistency was calculated using four different methods, and it was observed that the top four SNPs identified by GWAS for persistency as estimated by the DENND3, PSTPIP1, ADAMTS20, PRODH2, NPHS1, RREL2, FAM19A1, CLIC5, GPR12, DEFB125, STPG2, CFAP206, RINT1, EFCAB10, TCERG1, and C18H16orf78. Upon network enrichment, it was observed that TCERG1, RINT1, CLIC5, and PSTPIP1 are co-expressed with several other genes in the breast mammary tissue of human, indicating their possible role in persistency of lactation in dairy animals.In Canadian Holstein cattle, APC, LOC112448352, NDFIP2, LOC107131404, and PDP1. APC gene in the canonical WNT signaling pathway plays a critical role in the regulation of ovarian development. Mis-regulation of this key pathway in the adult ovary is associated with subfertility in mice. The APC gene has also been mapped to the QTL region for conception rate in Holstein cattle . The GRID2 gene is however reported to be associated with growth traits in Simmental cattle and may have role in regulating age at sexual maturity , the results obtained in the present study are bias free, as indicated by the FDR. The findings of the present study in the Murrah population will encourage researchers to come forward for GWAS in buffalo in a holistic manner.Optimum production and reproduction in buffaloes are long-standing questions in the Indian subcontinent. We present the analysis and identification of genomic regions that play role in shaping the selection and breeding decisions of Murrah buffalo for persistency of production and fertility. The dataset information presented in the paper is also submitted so that it can be used to compare and evaluate other breeds of buffalo based on the genomic information generated. The putative identified regions have a potential to improve the existing breeding decisions in this important dairy germplasm.PRJEB47270 (https://wwwdev.ebi.ac.uk/eva/?eva-study=PRJEB47270).The data presented in the study are deposited in the European Variation Archive repository, accession number The animal study was reviewed and approved by the ICAR-National Dairy Research Institute IAEC.VV conceptualized the work and interpreted the data. SC, GG, RA, and AM performed the collection of materials, and performed the data analysis. AV and SD provided the data and resources. VV and SC did writing of the manuscript and its critical evaluation. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.