Impact of Limosilactobacillus fermentum probiotic treatment on gut microbiota composition in sahiwal calves with rotavirus diarrhea: A 16S metagenomic analysis study".

BACKGROUND: Diarrhea poses a major threat to bovine calves leading to mortality and economic losses. Among the causes of calf diarrhea, bovine rotavirus is a major etiological agent and may result in dysbiosis of gut microbiota. The current study was designed to investigate the effect of probiotic Limosilactobacillus fermentum (Accession No.OR504458) on the microbial composition of rotavirus-infected calves using 16S metagenomic analysis technique. Screening of rotavirus infection in calves below one month of age was done through clinical signs and Reverse Transcriptase PCR. The healthy calves (n = 10) were taken as control while the infected calves (n = 10) before treatment was designated as diarrheal group were treated with Probiotic for 5 days. All the calves were screened for the presence of rotavirus infection on each day and fecal scoring was done to assess the fecal consistency. Infected calves after treatment were designated as recovered group. Fecal samples from healthy, recovered and diarrheal (infected calves before sampling) were processed for DNA extraction while four samples from each group were processed for 16S metagenomic analysis using Illumina sequencing technique and analyzed via QIIME 2. RESULTS: The results show that Firmicutes were more abundant in the healthy and recovered group than in the diarrheal group. At the same time Proteobacteria was higher in abundance in the diarrheal group. Order Oscillospirales dominated healthy and recovered calves and Enterobacterials dominated the diarrheal group. Alpha diversity indices show that diversity indices based on richness were higher in the healthy group and lower in the diarrheal group while a mixed pattern of clustering between diarrheal and recovered groups samples in PCA plots based on beta diversity indices was observed. CONCLUSION: It is concluded that probiotic Limosilactobacillus Fermentum N-30 ameliorate the dysbiosis caused by rotavirus diarrhea and may be used to prevent diarrhea in pre-weaned calves after further exploration.

Milk microbiota of Holstein Friesian cattle reared in Lahore: Association with mastitis.

The dairy industry is reshaping itself and becoming commercialized in Pakistan due to the increased demand for milk to overcome the shortage. Exotic breeds such as Holstein Friesian, a high milk producing breed have started being reared more on farms in Pakistan. Along with other issues, mastitis does affects the milk production of this breed. The objective of this study was to evaluate the milk composition in terms of bacterial communities in Holstein Friesian reared in Punjab, Pakistan and alteration in microbial composition with healthy and mastitic udder. Milk samples (n = 36) from farms rearing Holstein Friesian were collected. Among these samples, 05 samples from each three groups, HHF(healthy), CHF (clinical mastitis) and SHF (subclinical mastitis), based on their udder health condition, were processed using the 16 S r=RNA gene based technique. Diversity assessment as carried out by alpha diversity indices showed that milk samples from the udder infected with clinical mastitis were the least diverse and those from the healthy udder were more diverse. Beta diversity across samples showed a scattered pattern suggesting overlap amongst bacterial communities across different groups samples as depicted by PCA plots of beta diversity indices. The taxonomic profile revealed that Proteobacteria Firmicutes, Bacteroidota and Actinobacteriota were the major phyla detected across all groups. Proteobacteria dominated the HHF and SHF group while abundance of Firmicutes was higher in CHF group. Differences at other levels including order, genus and species were also recorded. The overall picture concludes that diverse microbiota is associated with different udder health conditions.

Mapping Genetic Markers Associated with Antigenicity and Host Range in H9N2 Influenza A Viruses Infecting Poultry in Pakistan.

The aim of the current study was to map the genetic diversity in the haemagglutinin (HA) glycoprotein of influenza A viruses (IAVs) of the H9N2 subtype. Twenty-five H9N2 IAVs were isolated from broiler chickens from March to July 2019. The HA gene was amplified, and phylogenetic analysis was performed to determine the evolutionary relationship. Important antigenic amino acid residues of HA attributed to immune escape and zoonotic potential were compared among H9N2 IAVs. Phylogenetic analysis revealed that sublineage B2 under the G1 lineage in Pakistan was found to be diversified, and newly sequenced H9N2 isolates were nested into two clades (A and B). Mutations linked to the antigenic variation and potential immune escape were observed as G72E (1/25, 4%), A180T (3/25, 12%), and A180V (1/25, 4%). A twofold significant reduction (P < 0.01) in log2 hemagglutination inhibition titers was observed with H9N2 IAV naturally harboring amino acid V180 instead of A180 in HA protein. Moreover, in the last 20 years, complete substitution at residues (T127D, D135N, and L150N) and partial substitution at residues (72, 74, 131, 148, 180, 183, 188, 216, 217, and 249, mature H9 HA numbering) associated with changes in antigenicity were observed. The presence of L216 in all H9N2 IAV isolates and T/V180 in four isolates in the receptor-binding site reveals the potential of these viruses to cross the species barrier to infect human or mammals. The current study observed the circulation of antigenically diverse H9N2 IAV variants that possess potential mutations that can escape the host immune system.

Nota de investigación- Mapeo de marcadores genéticos asociados con la antigenicidad y el rango de huéspedes en los virus de la influenza tipo A subtipo H9N2 que infectan a la avicultura en Pakistán. El objetivo del presente estudio fue mapear la diversidad genética en la glicoproteína hemaglutinina (HA) de los virus de la influenza A (IAV) del subtipo H9N2. Se aislaron veinticinco virus de influenza H9N2 de pollos de engorde de marzo a julio del 2019. Se amplificó el gene HA y se realizó un análisis filogenético para determinar la relación evolutiva. Se compararon importantes residuos de aminoácidos antigénicos de la hemaglutinina atribuidos al escape inmunológico y al potencial zoonótico entre los virus de la influenza aviar H9N2. El análisis filogenético reveló que el sublinaje B2 bajo el linaje G1 en Pakistán estaba diversificado, y los aislados de H9N2 recién secuenciados se agruparon en dos clados (A y B). Se observaron mutaciones relacionadas con la variación antigénica y el posible escape inmunológico como los residuos de aminoácidos G72E (1/25, 4%), A180T (3/25, 12%) y A180V (1/25, 4%). Se observó una reducción significativa al doble (P < 0.01) en los títulos de inhibición de la hemaglutinación log2 cuando el virus de la influenza aviar H9N2 albergaba naturalmente el aminoácido V180 en lugar del A180 en la proteína HA. Además, en los últimos 20 años, sustitución completa en los residuos (T127D, D135N y L150N) y sustitución parcial en los residuos (72, 74, 131, 148, 180, 183, 188, 216, 217 y 249, de acuerdo con la numeración de la HA subtipo madura) asociados con cambios en la antigenicidad. La presencia del residuo L216 en todos los aislados de influenza aviar H9N2 y T/V180 en cuatro aislados en el sitio de unión al receptor revela el potencial de estos virus para cruzar la barrera de las especies para infectar a humanos o mamíferos. El estudio actual observó la circulación de variantes antigénicamente diversas del virus de influenza aviar H9N2 que poseen mutaciones potenciales que pueden escapar del sistema inmunológico del huésped.

One Health Assessment of Bacillus anthracis Incidence and Detection in Anthrax-Endemic Areas of Pakistan.

Anthrax, a severe zoonotic disease, is infrequently reported in anthrax-endemic regions of Pakistan. Despite clinical reports indicating its presence, particularly cutaneous anthrax, there is insufficient laboratory evidence regarding disease occurrence and environmental persistence. The present study aimed to confirm Bacillus anthracis presence, accountable for animal mortality and human infection, while exploring environmental transmission factors. Between March 2019 and July 2021, a total of 19 outbreaks were documented. Of these, 11 affected sheep/goats in Zhob district and 8 affected cattle/sheep in Bajour Agency. Clinical signs suggestive of Bacillus anthracis outbreak were observed in 11 animals. Blood and swab samples were collected for confirmation. The study followed a One Health approach, analyzing animal, environmental (soil/plant), and human samples. Of the 19 outbreaks, 11 were confirmed positive for anthrax based on growth characteristics, colony morphology, and PCR. Soil and plant root samples from the outbreak areas were collected and analyzed microscopically and molecularly. Cutaneous anthrax was observed in six humans, and swab samples were taken from the lesions. Human serum samples (n = 156) were tested for IgG antibodies against PA toxin and quantitative analysis of anthrax toxin receptor 1 (ANTXR1). Bacillus anthracis was detected in 65 out of 570 (11.40%) soil samples and 19 out of 190 (10%) plant root samples from the outbreak areas. Four out of six human samples from cutaneous anthrax lesions tested positive for Bacillus anthracis. Human anthrax seroprevalence was found to be 11% and 9% in two districts, with the highest rates among butchers and meat consumers. The highest ANTXR1 levels were observed in butchers, followed by meat consumers, farm employees, meat vendors, veterinarians, and farm owners. These findings highlight the persistence of anthrax in the region and emphasize the potential public health risks.

Investigation of milk microbiota of healthy and mastitic Sahiwal cattle.

BACKGROUND: Sahiwal cattle is an indigenous cattle breed of Pakistan and mastitis is one of the major problems faced by Sahiwal cattle which hinders its production potential. The study was designed to investigate the milk microbiota of healthy and mastitic Sahiwal cattle as part of a multistep project to develop probiotics for the mitigation and control of mastitis. Milk samples of Sahiwal cattle (healthy clinical mastitis and subclinical mastitis) reared under similar husbandry and management practices were processed for 16S rRNA gene base metagenomics analysis. RESULTS: Results revealed that Proteobacteria were dominant in the healthy group and subclinical mastitis group (56.48% and 48.77%, respectively) as compared to the clinical mastitis group (2.68%). In contrast, Firmicutes were abundant in the clinical mastitis group (64%) as compared to the healthy and subclinical mastitis groups (15.87% and 38.98%, respectively). Dominant species assigned in the healthy group were Ignavibacterium album, Novosphingobium capsulatum, Akkermansia muciniphila and Lactobacillus fermentum.The clinical mastitis group was dominated by Streptococcus dysgalactiae and Corynebacterium bovis, while subclinical mastitis group included Lactobacillus fermentum and uncultured acidobacteriales and Akkermansia muciniphila as dominant species. Alpha diversity indices showed higher microbial diversity in the healthy group compared to the clinical and sub-clinical mastitis groups. CONCLUSION: It is concluded that the milk microbiota of healthy sahiwal cattle has higher diversity and dominant taxa in the different groups may be used as signature microbes for mastitis susceptibility. Akkermansia muciniphila is one of candidate specie that was identified and may be used for development of probiotics.

Molecular identification and infection pathology of Mycobacterium spp. in captive wild animals in Pakistan.

INTRODUCTION: Animal tuberculosis is an infectious, chronic, granulomatous, and debilitating disease affecting animals as well as humans. However, in recent decades, there have been many endemic geographic localities where animal tuberculosis has been identified in wildlife reservoirs, limiting the eradication program in cattle. This study aimed to identify animal tuberculosis in captive zoo animals in Pakistan. METHODOLOGY: In total, 185 morbid zoo animals were brought for postmortem examination at a veterinary postmortem facility. During the macroscopic examination, these animals were thoroughly examined for the presence of suggestive gross lesions of animal tuberculosis (granulomas/tubercles), and the pattern and distribution of these lesions in different organs. The Ziehl-Neelsen (ZN) staining was performed on smears prepared from granulomatous lesions of lung tissue followed by molecular identification of M. bovis and M. tuberculosis DNA using polymerase chain reaction (PCR). RESULTS: The postmortem examination revealed that 8.1% (15/185) of animals had gross tuberculosis lesions on the lungs and lymph nodes. The ZN staining of tissue smears showed 5.40% positivity while M. bovis and M. tuberculosis DNA was identified in 3.78 % and 1.1% of investigated animals, respectively. CONCLUSIONS: The study showed that animal tuberculosis is prevalent among wildlife in Pakistan and it may pose serious public health concerns to the people visiting these zoos and wildlife parks.

Genomic Diversity and Evolution of SARS-CoV-2 Lineages in Pakistan.

The emergence of SARS-CoV-2 variants has posed a challenge to disease control efforts worldwide. This study explored the genomic diversity and phylogenetic relationship of SARS-CoV-2 variants reported in Pakistan. Our objective was to understand the transmission dynamics of different lineages within the country. We retrieved and analyzed spike protein sequences from Pakistan and compared them with reference sequences reported worldwide. Our analysis revealed the clustering of Pakistan-origin isolates in nine different clades representing different regions worldwide, suggesting the transmission of multiple lineages within the country. We found 96 PANGO lineages of SARS-CoV-2 in Pakistan, and 64 of these corresponded to 4 WHO-designated variants: Alpha, Beta, Delta, and Omicron. The most dominant variants in Pakistan were Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2, AY.108), and Omicron (BA.2.75, BA.5.2), and the N-terminal domain and receptor binding regions were the most hypervariable regions of the spike gene. Compared to the reference strain, characteristic substitutions were found in dominant variants. Our findings emphasize the importance of continuously monitoring and assessing nucleotide and residue substitutions over time to understand virus evolutionary trends better and devise effective disease control interventions.

Exploring the Milk Microbiota of Healthy and Mastitic Nili Ravi Buffalo Using 16S rRNA Gene Base Metagenomic Analysis.

The Nili Ravi, a buffalo breed from Pakistan, significantly contributes to the dairy industry. Mastitis is one of the major challenges affecting milk production in this breed. The objective of the current study was to identify the bacterial communities and diversity in healthy and mastitic milk of this breed. Milk samples (n = 14) were collected from Nili Ravi buffaloes with different udder health statuses, i.e., healthy (5), subclinical mastitis (4), and clinical mastitis (5). The DNAs were extracted, subjected to partial amplification of 16S rDNA (V3 and V4 regions), and sequenced using the Illumina platform. The results revealed variations in the bacterial communities in the milk of animals with different udder health statuses. Proteobacteria was the predominant phylum in the healthy group, while clinical and subclinical mastitis milk had a higher abundance of Firmicutes. Dominant bacterial genera in the healthy group were Streptococcus (11.60%), Herbaspirillum (7.65%), and Staphylococcus (4.70%), whereas the clinical mastitis group was dominated by Streptococcus (33.96%), Staphylococcus (7.87%), and Corynebacterium (2.68%), and the subclinical mastitis group was dominated by Bacillus (15.70%), Corynebacterium (6.70%), and Staphylococcus (6.58%). Assignment of operational taxonomic units at the species level resulted in most species being assigned to uncultured or unknown bacteria or remaining unassigned. Alpha diversity indices indicated lower microbial diversity in the clinical mastitis group, while beta diversity indices showed a scattered pattern of sample clustering in PCA plots among different groups. It is concluded that bacterial diversity in the milk of Nili Ravi buffaloes suffering from clinical mastitis is lower compared to healthy and subclinical mastitis cases. It is concluded that the variations in the microbiota of healthy and mastitic milk may be further investigated and exploited as signature microbes associated with the udder health status of Nili Ravi buffalo.

Temporal study of wastewater surveillance from September 2020 to March 2021: an estimation of COVID-19 patients in Lahore, Pakistan.

The first aim of study was to quantify the viral load in the wastewater samples by RT-qPCR testing in Lahore population to estimate the number of patients affected and predict the next resurgence of COVID-19 wave in the city. The second aim of the study was to determine the hotspot areas of Lahore which remained positive more often for virus with high viral load. In this study, n = 420 sewage samples were collected on an average of two weeks intervals from 30 different sewage water disposal stations (14 sampling events) from Sept 2020 to March 2021. RNA was extracted and quantified by RT-qPCR without concentrating the virus in samples. Number of positive disposal sites (7-93%), viral load from sewage samples (100.296 to 103.034), and estimated patients (660-17,030) ranged from low to high according to the surge and restrain of 2nd and 3rd COVID-19 waves in the country. The viral load and estimated patients were reported high in January 2021 and March 2021 which were similar to the peak of 2nd and 3rd waves in Pakistan. Site 18 (Niaz Baig village DS) showed the highest viral load among all sites. Findings of the present study helped to estimate the number of patients and track the resurgence in COVID-19 waves in Lahore particularly, and in Punjab generally. Furthermore, it emphasizes the role of wastewater-based epidemiology to help policymakers strengthen the quarantine measures along with immunization to overcome enteric viral diseases. Local and national stake holders should work in collaboration to improve the environmental hygiene to control the disease.

Genomic architecture and evolutionary relationship of BA.2.75: A Centaurus subvariant of Omicron SARS-CoV-2.

In this study, we explored the genomic architecture and phylogenomic relationship of BA.2.75, a subvariant of Omicron SARS-CoV-2. A set of 1468 whole-genome sequences of BA.2.75, submitted by 28 countries worldwide were retrieved from GISAID and used for finding genomic mutations. Moreover, the phylogenetic analysis of BA.2.75 was performed by using 2948 whole-genome sequences of all sub-variants of Omicron along with the Delta variant of SAS-CoV-2. We detected 1885 mutations, which were further grouped into 1025 missense mutations, 740 silent mutations, 72 mutations in non-coding regions, 16 in-frame deletions, 02 in-frame insertions, 8 frameshift deletions, 8 frameshift insertions and 14 stop-gained variants. Additionally, we also found 11 characteristic mutations having a prevalence of 81-99% and were not observed in any of the previously reported variant of SARS-CoV-2. Out of these mutations K147E, W152R, F157L, E210V, V213G, G339H were found in the NTD, and G446S & N460K in the RBD region of the Spike protein, whereas S403L and T11A were present in the NSP3, and E protein respectively. The phylogenetic relationship of this variant revealed that BA.2.75 is descended from the Omicron sub-variant BA.5. This evolutionary relationship suggests that the surge of BA.5 infections can reduce the severity of the infections accredited to BA.2.75. These findings would also improve our knowledge and understanding that how genetic similarities in different variants of SARS-CoV-2 can prime the immune system to fight off the infection caused by one subvariant, after defeating the other.

Molecular Characterization and Selection of Indigenous SARS-CoV-2 Delta Variant for the Development of the First Inactivated SARS-CoV-2 Vaccine of Pakistan.

Vaccines are one of the efficient means available so far for preventing and controlling the infection rate of COVID-19. Several researchers have focused on the whole virus's (SARS-CoV-2) inactivated vaccines which are economically efficient to produce. In Pakistan, multiple variants of SARS-CoV-2 have been reported since the start of the pandemic in February 2020. Due to the continuous evolution of the virus and economic recessions, the present study was designed to develop an indigenous inactivated SARS-CoV-2 vaccine that might help not only to prevent the COVID-19 in Pakistan, it will also save the country's economic resources. The SARS-CoV-2 were isolated and characterized using the Vero-E6 cell culture system. The seed selection was carried out using cross-neutralization assay and phylogenetic analysis. The selected isolate of SARS-CoV-2 (hCoV-19/Pakistan/UHSPK3-UVAS268/2021) was inactivated using beta-propiolactone followed by vaccine formulation using Alum adjuvant, keeping the S protein concentration as 5 µg/dose. The vaccine efficacy was evaluated by in vivo immunogenicity testing in laboratory animals and in in vitro microneutralization test. The phylogenetic analysis revealed that all the SARS-CoV-2 isolates reported from Pakistan nested into different clades, representing multiple introductions of the virus into Pakistan. The antisera raised against various isolates from different waves in Pakistan showed a varied level of neutralization titers. However, the antisera produced against a variant (hCoV-19/Pakistan/UHSPK3-UVAS268/2021; fourth wave) efficiently neutralized (1:64-1:512) all the tested SARS-CoV-2 isolates. The inactivated whole virus vaccine of SARS-CoV-2 was safe and it also elicited a protective immune response in rabbits and rhesus macaques on the 35th-day post-vaccination. The activity of neutralizing antibodies of vaccinated animals was found at 1:256-1:1024 at 35 days post-vaccination, indicating the effectiveness of the double-dose regime of the indigenous SARS-CoV-2 vaccine.

Mucormycosis an added burden to Covid-19 Patients: An in-depth systematic review.

As of 25th July, 2022, global Disease burden of 575,430,244 confirmed cases and over 6,403,511 deaths have been attributed to coronavirus disease 2019 (COVID-19). Co-infections/secondary infections continue to plague patients around the world as result of the co-morbidities like diabetes mellitus, biochemical changes caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) especially significant elevation in free iron levels, immune suppression caused by SARS-CoV-2, and indiscriminate use of systemic corticosteroids for the treatment of severe COVID-19 disease. In such circumstances, opportunistic fungal infections pose significant challenge for COVID-19 disease therapy in patients with other co-morbidities. Although COVID-19-associated Mucormycosis (CAM) has been widely recognized, currently extensive research is being conducted on mucormycosis. It has been widely agreed that patients undergoing corticosteroid therapy are highly susceptible for CAM, henceforth high index of screening and intensive care and management is need of an hour in order to have favorable outcomes in these patients. Diagnosis in such cases is often delayed and eventually the disease progresses quickly which poses added burden to clinician and increases patient load in critical care units of hospitals. A vast perusal of literature indicated that patients with diabetes mellitus and those with other co-morbidities might be highly vulnerable to develop mucormycosis. In the present work, the case series of three patients presented at Chest Disease Hospital Srinagar, Jammu and Kashmir infected with CAM has been described with their epidemiological data in supplementary section. All these cases were found to be affected with co-morbidity of Diabetes Mellitus (DM) and were under corticosteroid therapy. Furthermore, given the significant death rate linked with mucormycosis and the growing understanding of the diseases significance, systematic review of the literature on CAM has been discussed and we have attempted to discuss emerging CAM and related aspects of the disease.

Renessans Helps in Early Clearance of SARS-CoV-2: In-Vivo Activity of the Iodine Complex in Rhesus macaque.

Iodine complexes have known antimicrobial properties along with reported in-vitro antiviral activity for several viruses. Renessans is one such product with iodine complexes and ascorbic acid. The present study was designed to determine its efficacy for SARS-CoV-2 in Rhesus macaque. Rhesus macaque were assigned to: A) prophylactic group (n = 3), (B) treatment group (n = 3), (C) infection control group (n = 4), and (D) negative control group (n = 4). Groups A, B, and C were challenged with 2 × 106 TCID of SARS-CoV-2. The prophylactic group (A) was administered Renessans from 5 days before infection till 8 days postinfection (DPI). The treatment group (B) was administered Renessans from 3 till 8 DPI. Group C was administered water-insoluble fractions only. Nasal swabs from all monkeys of groups A, B, and C remained positive for SARS-CoV-2 till 2 and 7 DPI, while the swabs became negative for groups A and B at 14 DPI. Likewise, fecal matter of monkeys in group A returned negative results during the experiment, while that of group B had significantly decreased viral load (101.5 genome copies/mL) compared to group C (103 genome copies/mL). Hence, it is concluded that Renessans has in-vivo SARS-CoV-2 activity and may result in early clearance of SARS-CoV-2.

Activity of plant essential oils against antibiotic resistant Enterococcus faecalis isolated from diarrheic children.

Activity of plant essential oils and their fractions was evaluated against characterized isolates of antibiotic resistant Enterococcus faecalis recovered from diarrheic children. The isolates were confirmed by polymerase chain reaction (PCR) targeting 16S rRNA gene amplification followed by nucleotide sequencing and accession numbers retrieved were MW349990.1, MW349859.1, MW332122.1, MW356805.1, MW349975.1, MW349988.1, MW356790.1, MW356244.1, MW341593.1 and MW332549.1. These isolates were screened for antibiotic susceptibility to a wide range of antibiotic groups and mean zone of inhibition (ZOI) of all antibiotics were recorded. Antibacterial activity of plant essential oils (n=05) was checked against three antibiotic resistant isolates of E. faecalis. Three plant essential oils having higher ZOI including Cinnamomum verum, Syzygium aromaticum and Nigella sativa were used against resistant E. faecalis isolates to determine minimum inhibitory concentration (MIC). The lowest MIC observed was of S. aromaticum (11.39±3.94 mg mL-1). The S. aromaticum n-hexane plus chloroform fraction displayed higher mean ZOI (16.67±2.51 mm), while the lowest MIC was of n-hexane oil fraction. Based upon gas chromatography-mass spectrometry (GC/MS) analysis, the most effective fatty acid was eugenic acid which is present in higher proportion in both fractions. These fractions of essential oils proved safe for the treatment of antibiotic resistant diarrheic cases of children caused by E. faecalis.

Rate of Multiple Viral and Bacterial CoInfection(s) in Influenza A/H9N2-Infected Broiler Flocks.

Repeated cases of low pathogenic influenza A/H9N2 virus (IAV/H9N2) have been reported in commercial chickens since its emergence in 1998 in Pakistan. However, recently increased mortality and severe respiratory complications under field conditions have been noticed, suggesting concomitant influenza infections with respiratory viral and/or bacterial pathogens. Therefore, the present study aimed to investigate the presence of IAV/H9N2 coinfecting with multiple viral and bacterial pathogens in broiler chicken flocks. We surveyed 60 broiler flocks with respiratory signs from March through July 2019 in Punjab, Pakistan. Suspected flocks were screened for the presence of IAV using a lateral-flow device. Tracheal, cloacal, and bone marrow samples were collected and further tested for seven viral agents (chicken anemia; Newcastle disease; infectious bronchitis; infectious laryngeotracheitis [ILT]; and IAV subtypes H9, H7, and H5) and three bacterial agents (Mycoplasma gallisepticum; Mycoplasma synovae; Ornithobacterium rhinotracheale [ORT]) using PCR assays. Upon initial screening for IAV, 35/60 (58.3%) flocks tested positive. The coinfection of IAV/H9N2 with other pathogens was detected in 25 (71.4%) flocks and only IAV/H9N2 was detected in 10 (28.6%) flocks out of total positive IAV flocks (n = 35). IAV subtypes H5 and H7, ILT, and ORT were not detected throughout the study period. The detection rate of double, triple, and quadruple combinations of coinfections with IAV/H9N2 were 37% (13 flocks), 26% (9 flocks), 9% (3 flocks), respectively. Higher average mortality (28.5%) was found in broiler chicken flocks coinfected with viral and/or bacterial pathogens than in flocks where only H9 low pathogenic IAV/H9N2 was detected (20.8%). In conclusion, higher circulation of IAV/H9N2 with other viral and bacterial pathogens may contribute to higher production and economic losses at the farm level.

Nota de investigación- Tasa de coinfecciones virales y bacterianas múltiples en parvadas de pollos de engorde infectadas con virus influenza A/H9N2. Se han reportado varios casos del virus de influenza A de baja patogenicidad H9N2 (IAV/H9N2) en pollos comerciales desde su aparición en 1998 en Pakistán. Sin embargo, recientemente se ha observado un aumento de la mortalidad y complicaciones respiratorias graves en condiciones de campo, lo que sugiere infecciones concomitantes de influenza con patógenos respiratorios virales y/o bacterianos. Por lo tanto, el presente estudio tuvo como objetivo investigar la presencia del virus de influenza aviar H9N2 coinfectando con múltiples patógenos virales y bacterianos en parvadas de pollos de engorde. Se evaluaron 60 parvadas de pollos de engorde con signos respiratorios desde marzo hasta julio del año 2019 en Punjab, Pakistán. Las parvadas sospechosas fueron analizadas para detectar la presencia del virus de influenza aviar utilizando un dispositivo de flujo lateral. Se recolectaron muestras traqueales, cloacales y de médula ósea y se analizaron para detectar siete agentes virales (anemia infecciosa aviar, enfermedad de Newcastle, bronquitis infecciosa, laringeotraqueítis infecciosa [ILT] y subtipos H9, H7 y H5 de influenza aviar) y tres agentes bacterianos (Mycoplasma gallisepticum ; Mycoplasma sinovae; Ornithobacterium rhinotracheale [ORT]) utilizando ensayos de PCR. Tras la detección inicial del virus de la influenza aviar, 35/60 (58.3 %) parvadas resultaron positivas. La coinfección del virus de la influenza H9N2 con otros patógenos se detectó en 25 (71.4 %) parvadas y el virus de influenza aviar H9N2 fue detectado solo en 10 (28.6 %) parvadas del total de parvadas positivas (n = 35). Los subtipos H5 y H7 del virus de influenza, ILT y ORT no se detectaron durante el período de estudio. La tasa de detección de combinaciones dobles, triples y cuádruples de coinfecciones con el virus de influenza H9N2 fue del 37 % (13 parvadas), del 26% (9 parvadas), del 9 % (3 parvadas), respectivamente. Se encontró una mortalidad promedio más alta (28.5 %) en lotes de pollos de engorde coinfectados con patógenos virales y/o bacterianos que en lotes donde solo se detectó al virus de influenza H9 de baja patogenicidad (20.8%). En conclusión, una mayor circulación del virus de influenza aviar H9N2 con otros patógenos virales y bacterianos puede contribuir a mayores pérdidas en la producción y económicas a nivel de granja.

Integration of RT-LAMP and Microfluidic Technology for Detection of SARS-CoV-2 in Wastewater as an Advanced Point-of-Care Platform.

Development of lab-on-a-chip (LOC) system based on integration of reverse transcription loop-mediated isothermal amplification (RT-LAMP) and microfluidic technology is expected to speed up SARS-CoV-2 diagnostics allowing early intervention. In the current work, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) and RT-LAMP assays were performed on extracted RNA of seven wastewater samples from COVID-19 hotspots. RT­LAMP assay was also performed on wastewater samples without RNA extraction. Current detection of SARS-CoV-2 is mainly by RT-qPCR of ORF (ORF1ab) and N genes so we targeted both to find the best target gene for SARS-CoV-2 detection. We also performed RT-LAMP with/without RNA extraction inside microfluidic device to target both genes. Positivity rates of RT-qPCR and RT-LAMP performed on extracted RNA were 100.0% (7/7) and 85.7% (6/7), respectively. RT-qPCR results revealed that all 7 wastewater samples were positive for N gene (Ct range 37-39), and negative for ORF1ab, suggesting that N gene could be the best target gene for SARS-CoV-2 detection. RT-LAMP of N and ORF (ORF1a) genes performed on wastewater samples without RNA extraction indicated that all 7 samples remains pink (negative). The color remains pink in all microchannels except microchannels which subjected to RT-LAMP for targeting N region after RNA extraction (yellow color) in 6 out of 7 samples. This study shows that SARS-CoV-2 was successfully detected from wastewater samples using RT-LAMP in microfluidic chips. This study brings the novelty involving the use of wastewater samples for detection of SARS-CoV-2 without previous virus concentration and with/without RNA extraction.

The transmission of human mitochondrial DNA in four-generation pedigrees.

Most of the pathogenic variants in mitochondrial DNA (mtDNA) exist in a heteroplasmic state (coexistence of mutant and wild-type mtDNA). Understanding how mtDNA is transmitted is crucial for predicting mitochondrial disease risk. Previous studies were based mainly on two-generation pedigree data, which are limited by the randomness in a single transmission. In this study, we analyzed the transmission of heteroplasmies in 16 four-generation families. First, we found that 57.8% of the variants in the great grandmother were transmitted to the fourth generation. The direction and magnitude of the frequency change during transmission appeared to be random. Moreover, no consistent correlation was identified between the frequency changes among the continuous transmissions, suggesting that most variants were functionally neutral or mildly deleterious and thus not subject to strong natural selection. Additionally, we found that the frequency of one nonsynonymous variant (m.15773G>A) showed a consistent increase in one family, suggesting that this variant may confer a fitness advantage to the mitochondrion/cell. We also estimated the effective bottleneck size during transmission to be 21-71. In summary, our study demonstrates the advantages of multigeneration data for studying the transmission of mtDNA for shedding new light on the dynamics of the mutation frequency in successive generations.

CRISPR-Cas System: An Adaptive Immune System's Association with Antibiotic Resistance in Salmonella enterica Serovar Enteritidis.

Several factors are involved in the emergence of antibiotic-resistant bacteria and pose a serious threat to public health safety. Among them, clustered regularly interspaced short palindromic repeat- (CRISPR-) Cas system, an adaptive immune system, is thought to be involved in the development of antibiotic resistance in bacteria. The current study was aimed at determining not only the presence of antibiotic resistance and CRISPR-Cas system but also their association with each other in Salmonella enteritidis isolated from the commercial poultry. A total of 139 samples were collected from poultry birds sold at the live bird markets of Lahore City, and both phenotypic and genotypic methods were used to determine antimicrobial resistance. The presence of the CRISPR-Cas system was determined by PCR, followed by sequencing. All isolates of S. enteritidis (100%) were resistant to nalidixic acid, whereas 95% of isolates were resistant to ampicillin. Five multidrug-resistant isolates (MDR) such as S. enteritidis isolate (S. E1, S. E2, S. E4, S. E5, and S. E8) were found in the present study. The CRISPR-Cas system was detected in all of these MDR isolates, and eight spacers were detected within the CRISPR array. In addition, an increased expression of CRISPR-related genes was observed in the standard strain and MDR S. enteritidis isolates. The association of the CRISPSR-Cas system with multiple drug resistance highlights the exogenous acquisition of genes by horizontal transfer. The information could be used further to combat antibiotic resistance in pathogens like Salmonella.

A Cross-Sectional Survey of Knowledge, Attitude, and Practices of University Students in Pakistan Regarding COVID-19.

The COVID-19 pandemic is striking the world with serious public health and socioeconomic complications. The pandemic has influenced all forms of daily life, including educational institutions. Therefore, this cross-sectional survey was conducted to understand knowledge, attitudes, and practices related to COVID-19 among the students of the University of Veterinary and Animal Sciences, Lahore. The data was collected using an online self-directed questionnaire. The survey form includes six items about sociodemographic characteristics, 14 knowledge-based questions, seven questions on attitude, and eight questions on practices. The sample number was calculated using the Raosoft sample size calculator. A total number of 3,854 students, including 1,823 men and 2,031 women, were engaged in this survey, having student representation from all the provinces in the country. The data were analyzed using a chi-square test. A total of 97% of the students knew that the etiological agent of COVID-19 is a virus and that it is a disease of the respiratory system (94%). Many students kept visiting their relatives during the lockdown (45%), and their relatives kept visiting them at home (59%). The responses from the students varied a lot on specific questions about the transmission of the virus. Women tended to have less information regarding precautionary travel measures (p < 0.01), but supplemental knowledge of prevention of disease transmission from positive patients (p < 0.01). Conclusively, the majority of the university students surveyed had imperative knowledge, a good attitude, and active practice in response to the COVID-19 outbreak. Moreover, the KAP scores have varied by demography, gender, and the number of family members. Therefore, continuous awareness of preventative behaviors should be disseminated regularly in emergencies.