Assessment of expected breeding value for milk production traits of Murrah buffaloes under different heat stress zones in Indian sub-tropical climatic conditions.

Present study was conducted to investigate the impact of temperature humidity index (THI) on expected breeding value (EBV) for daily milk yield (DMY) and monthly test day fat% (MTDF%) and monthly test day SNF% (MTDSNF%) of Murrah buffaloes at ICAR-National Dairy Research Institute, Karnal. 302,101 records of DMY and 9864 records each on MTDF% and MTDSNF% buffaloes spanned over 20 years (1994 to 2013) from NDRI, Karnal, and meteorological were collected from ICAR-CSSRI, Karnal. The year was classified into three zones: non-heat stress (NHSZ), heat stress (HSZ), and critical heat stress zone (CHSZ) based on THI. The heritability (h2) estimates for DMY, MTDF%, and MTDSNF% in NHSZ were 0.432 ± 0.054, 0.090 ± 0.004, and 0.070 ± 0.002; in HSZ 0.491 ± 0.073, 0.112 ± 0.003, and 0.052 ± 0.001; and in CHSZ 0.524 ± 0.077, 0.116 ± 0.004, and 0.092 ± 0.003, respectively. The repeatability (r) for DMY, MTDF%, and MTDSNF% in NHSZ were 0.528 ± 0.006, 0.166 ± 0.007, and 0.135 ± 0.007; in HSZ 0.572 ± 0.007, 0.198 ± 0.006, and 0.077 ± 0.006; and in CHSZ 0.599 ± 0.008, 0.217 ± 0.004, and 0.156 ± 0.009, respectively. EBV for DMY and MTDF% was maximum in NHSZ (8.85 kg and 7.85%) and in HSZ (7.27 kg and7.78%) and lowest (6.90 kg and 7.77%) at CHSZ. For MTDSNF%, EBV was highest during NHSZ (9.6403), declined to 9.6265 at HSZ, and marginally elevated to 9.6271 at CHSZ. Impact of climatic factors on milk production and constituent traits is vital, and proper management should be followed during the heat stress and critical heat stress periods to improve the production performance of Murrah buffaloes.

Delay in puberty is dependent on heat shock protein B1 expression in native cross layers of Punjab under heat stress.

Small heat shock protein B1 (HspB1) has been reported to play an essential role in thermotolerance. This study aimed to determine a correlation (if any) between HspB1 expression and age at first egg lay (puberty) in a native cross-layer poultry of Punjab under heat stress. Forty native cross-layer birds were reared in two different seasons, viz. summer (THI was more than 27), classified as the heat-stressed group (n = 20) and winter season (THI was less than 21), classified as the control group (n = 20). Blood was collected from both the groups of birds in their 15th week of age and at puberty. Serum catalase and superoxide dismutase activities, reduced glutathione and corticosterone concentration and lipid peroxidation were measured to assess the oxidative stress in both the groups of birds. The serum antioxidants significantly decreased whilst corticosterone levels and lipid peroxidation significantly elevated in birds in response to summer heat stress. Moreover, in summer season, the activities of the antioxidant enzymes further decreased and lipid peroxidation further increased significantly in birds from their pre-pubertal stage to puberty, which was not observed during the winter season. A clone of chicken HspB1 in BL21 (DE3) cells was revived, and recombinant HspB1 was purified using Ni-NTA agarose column. Serum HspB1 concentration was estimated in different groups of birds by indirect ELISA that has been standardized using the recombinant chicken HspB1. Compared to the control, birds under heat stress had significantly higher serum HspB1 levels. The delay in puberty of all the heat-stressed birds was significantly associated with the increase in their serum HspB1 levels. Taken together, the expression of HspB1 was found to be associated with age at puberty in the native cross poultry layers of Punjab.

Determination of heat stress zone for daily milk yield using carryover heat effect model in Murrah buffaloes.

The objective of the present study was to determine heat stress zones (HSZ) in a year for daily milk yield (DMY) of Murrah buffaloes. Data for the study included 2,67,599 daily milk yield records of 445 Murrah buffaloes sired by 171 bulls as well as climatic parameters, viz. dry bulb temperature (Tdb) and wet bulb (Twb) temperatures spanning over 16 years (2001-2016). The regression analysis of DMY on carryover heat effect indicated that the decline in DMY was significant above the carryover heat effect (CHE) 70. April-November were critical for daily milk yield, and maximum decline in daily milk yield was observed during the month of August in Murrah buffaloes. Therefore, two zones in a year were classified as non-heat stress zone (NHSZ) with CHE < 70 for months December-March and heat stress zone (HSZ) with CHE > 70 during April-November.

The overexpression of Hsp90B1 is associated with tumorigenesis of canine mammary glands.

Heat shock proteins (Hsp) are molecular chaperones that are responsible for protein folding and maintenance of cellular homeostasis. Hsp90, an important member of HSP family, has an important role in breast cancer. Glucose-regulated protein 94 (Grp94) is the endoplasmic reticulum paralog of Hsp90 encoded by Hsp90B1 gene. To test if this protein is overexpressed in dogs with mammary tumor, we estimated and compared its serum levels in healthy dogs and that of dogs with mammary tumors. Hsp90B1 mRNA expression was measured in tumorous and healthy mammary tissues (from age- and breed-matched dogs) by real-time PCR. The gene was found to be overexpressed in mammary tumors (3.586 ± 0.067 times). Further, it was heterologously expressed in a prokaryotic system as 90 kDa protein. A recombinant Grp94-based sandwich ELISA was developed to quantify serum Grp94 in dogs with mammary tumors. Based on receiver-operating characteristics' analysis, the assay was found to be 90.62% sensitive and 93.75% specific for a cutoff value of 0.35 with respect to histopathological staining in diagnosing the disease. The t test showed that serum Grp94 levels were significantly elevated (92.97 ± 3.62 ng/ml) in dogs with mammary tumors compared with healthy controls (10.30 ± 0.79 ng/ml) (p < 0.0001). These findings suggest that Grp94 might act as a potential biomarker for prognosis of canine mammary tumors and monitoring its therapy.

Associations of teat morphometric parameters and subclinical mastitis in riverine buffaloes.

The possible association between teat morphometric traits and subclinical mastitis (SCM) in dairy buffaloes was studied. Teat morphometric parameters, i.e. teat shape (bottle, conical, cylindrical, and others), teat-end shape (flat, round, and pointed), teat length (TL), teat diameter (TD), and teat-end to floor distance were measured before milking, but after proper milk let-down, in clinically healthy buffaloes (47 Murrah and 34 Nili-Ravi breeds). Subclinical mastitis was defined on the basis of bacteriology and somatic cell count (SCC) of quarter foremilk samples. A high proportion of cylindrical teats (40%) and pointed teat-ends (64·4%) was observed. Hind teats were longer and thicker than fore teats (P < 0·05). A significant breed effect was found with respect to teat shape, length and diameter (P < 0·05). Teats were mostly cylindrical (43·3 vs. 35·4%) and conical (34·2 vs. 30·8%) shaped, smaller (mean 8·2 vs. 9·5 cm) and thinner (mean 3·3 vs. 3·6 cm) in the Murrah breed compared with the Nili-Ravi breed. Teats that had 'other' shapes and were longer, wider, and placed closer to the floor were more associated with SCM (P < 0·05). Mean SCC was significantly higher (P < 0·05) in Nili-Ravi buffaloes, teat shapes classified as 'others', and quarters with SCM. Teat morphometric traits seem to be associated with indicators of udder health in buffaloes, thus, their inclusion in breeding programmes for selection against undesirable dairy type traits may be of value in reducing susceptibility to intramammary infections in Indian buffaloes.

Genetic analysis of persistency in HF crossbred cattle at an organized farm of northern India.

The present study was undertaken to estimate effect of various genetic and non-genetic factors on persistency of milk production and to identify the most appropriate persistency method that fits best in our environment. In the present study, effects of different non-genetic factors, viz. year, season, days to attain peak yield, and genetic group based on the level of exotic inheritance on persistency of milk yield in crossbred cattle were studied. Data comprised of 686 first lactation daily milk yield records of crossbred cattle that were maintained at GADVASU dairy farm over a period of 25 years from 1991 to 2015 were utilized to calculate persistency coefficients by four methods, viz., Ludwick and Peterson method (P1), Mahadevan method (P2), ratio method (P3), and Prasad et al. method (P4). Overall least squares means for persistency by Ludwick and Peterson method (P1), Mahadevan method (P2), ratio method (P3), and Prasad et al. method (P4) were 0.896 ± 0.096, 1.385 ± 0.224, 187.207 ± 26.398, and 0.621 ± 0.098, respectively. Effect of sires was significant (P < 0.05) on P2 and P4 methods. Effect of genetic group on all four methods was non-significant. Period of calving had significant (P < 0.01) effect on persistency of milk yield (P2, P3, and P4 methods). Effect of season of calving on persistency of milk yield was found to be significant in all estimates obtained by the four methods. Summer and autumn calvers were most persistent whereas spring and winter calvers were least persistent for (P2, P3, and P4 methods). Persistency of milk yield was significantly (P < 0.05) affected by days to attain peak yield in P1 and P2 methods. Maximum persistency was obtained in animals attaining peak at 41-57 days of lactation and minimum in < 41 days for Mahadevan method and ratio method. The highest heritability of persistency and minimum value of standard error was estimated as 0.275 ± 0.11 for the Mahadevan method followed by the Prasad method (0.197 ± 0.10) by half sib correlation method. The maximum coefficient of variation which indicates available variability was estimated as 20.788% for persistency by the Mahadevan method followed by 18.969% for the Prasad method. The highest correlation was also observed between P1 and P3 methods by Spearman's and Pearson's correlation for least squares breeding value of the sires. On the basis of heritability, standard error of heritability, and coefficient of variation, it can be concluded that the Mahadevan method followed by the Prasad method suits best to our environment for animals in first lactation as well as they can be utilized for effective selection for higher persistency in crossbred animals of Punjab.

Cellular thermotolerance is independent of HSF 1 expression in zebu and crossbred non-lactating cattle.

Heat stress is an important domain of research in livestock due to its negative impact on production and disease resistance. The augmentation of stress in the body stimulates the antioxidative activity comprising various enzymes (viz., catalase, superoxide dismutase), metabolites (reduced glutathione, etc.), vitamins, minerals, etc. to combat the situation. The major key players involved in regulation of heat shock response in eukaryotes are the transcription factors, called as heat shock factors (HSF). They activate the heat shock protein (HSP) genes by binding to their promoters. Lymphocytes are considered to be the best model to evaluate the immunity in any living body as it contains plethora of white blood cells (WBCs).In this study, the peripheral blood mononuclear cells (PBMC) obtained from non-lactating Sahiwal vis-à-vis crossbred (Holstein Friesian × Sahiwal) cattle with 75% or more exotic inheritance were subjected to heat shock at 39, 41, and 43 °C in three different incubators, in vitro. The cell count and viability test of pre and post heat stress of concerned PBMCs indicated that the crossbreeds are more prone to heat stress as compared to Sahiwal. The reverse transcription PCR (qRT-PCR) expression data revealed an increment in HSF1 expression at 41 °C which subsequently declined (non-significantly) at 43 °C in both breeds post 1 h heat shock. However, the association between the HSF 1 expression and antioxidative activity through correlation analysis was found to be non-significant (P < 0.05), though enzymatic activity appeared to behave in a similar fashion in both breeds at 5% level of significance (P < 0.05). This rule out the role of HSF1 expression level on the activity of enzymes involved in oxidative stress in vitro in zebu and crossbred cattle.

Effect of ß-hydroxybutyrate acid on gene expression levels of antioxidant biomarkers and growth hormone-related genes in liver cell culture.

Introduction: In dairy cattle, oxidative stress is a predominant problem associated with diseases and reproductive health issues. This study aimed to detect the variation in the antioxidant biomarkers by adding different concentrations of ß-hydroxybutyric acid (BHBA) and sought to elucidate its effects on the gene expression levels of growth hormone (GH) and antioxidant biomarkers in bovine hepatocytes. Material and Methods: Four antioxidant biomarkers, namely malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH Px) were evaluated using commercially available bovine ELISA kits. The expression levels of the bovine GH, its receptor (GHR), insulin-like growth factor (IGF), IGF-1, IGF-1 receptor, CAT, SOD, GSH-Px and ß-actin (as a reference) genes in liver cell culture were determined by reverse transcriptase-PCR assay. Results: With the increase of BHBA concentration and culture time, the activities of SOD, CAT, and GSH Px biomarkers in hepatocytes decreased. However, the content of MDA in hepatocytes increased gradually with the increase of hepatocyte culture time and BHBA concentration. The qPCR results revealed that after adding BHBA, gene expression levels of GSH-Px, SOD and IGF biomarkers in hepatocytes began to differ in the culture groups at 12 h, whereas the gene expression level of the CAT and GHR biomarkers in hepatocytes began to differ at 6 h. Conclusion: Quantitative PCR results showed that the BHBA significantly downregulated the expression levels of the GHR gene and CAT, GSH Px and SOD antioxidant biomarker genes.

A comprehensive review of genomic perspectives of canine diseases as a model to study human disorders.

The domestic dog has been given considerable attention as a system for investigating the genetics of human diseases. Population diversity and breed structure are unique features that make dogs particularly amenable to genetic studies. Dogs show distinguished features of breed-specific homogeneity, which is associated with striking interbreed heterogeneity. This review discusses the significance of studying the genetic maps, genome-wide association studies (GWAS), and usefulness of this species as an animal model. Most canine genetic disorders are similar to those of humans, including inherited, psychiatric, and genetic disorders. In addition to revealing new candidate genes, canine models allow access to experimental resources, such as cells, tissues, and even live animals, for research and intervention purposes.

Le chien domestique a reçu une attention considérable en tant que système d'investigation de la génétique des maladies humaines. La diversité de la population et la structure de la race sont des caractéristiques uniques qui rendent les chiens particulièrement propices aux études génétiques. Les chiens présentent des caractéristiques distinctes d'homogénéité spécifique à la race, qui est associée à une hétérogénéité interraciale frappante. Cette revue traite de l'importance de l'étude des cartes génétiques, des études d'association à l'échelle du génome (GWAS) et de l'utilité de cette espèce en tant que modèle animal. La plupart des troubles génétiques canins sont similaires à ceux des humains, y compris les troubles héréditaires, psychiatriques et génétiques. En plus de révéler de nouveaux gènes candidats, les modèles canins permettent d'accéder à des ressources expérimentales, telles que des cellules, des tissus et même des animaux vivants, à des fins de recherche et d'intervention.(Traduit par Docteur Serge Messier).

Molecular epidemiology, phylogenetic analysis and risk assessment of Anaplasma marginale from naturally infected bovines of Punjab (India).

Anaplasmosis, an infectious vector borne rickettsial disease caused by different species of Anaplasma transmitted through mechanical and biological (tick) processes has a great concern to livestock industry due to its associated economic losses. The current cross sectional comprehensive epidemiological study was conducted from August 2020 to November 2021 on 391 (277 cattle and 114 buffaloes) bovines from different districts of five agro climatic zones of Punjab state. Classical microscopic examination of Romanowsky stained blood smears showed an occurrence of 13.00%, while DNA amplification targeting major surface protein (msp5) of Anaplasma marginale revealed the 382 bp amplicon in 32.48% samples. Zone based molecular prevalence of A. marginale was highest in the Undulating zone (41.93%) and least in the Sub-Mountain zone (18.84%). The prevalence in Central plain zone, Western plain zone, and Western zone were 40.15, 30.95 and 29.91%, respectively. An overall molecular prevalence of A. marginale was 34.52, 32.75 and 20.0% in young, adults and calves, respectively, the difference being non-significant. Anaplsma marginale was more prevalent in unorganized farms (38.22%; 60/157) than organized farms (28.63%; 66/234). Risk factors analysis revealed young, female cattle at unorganized farms were more prone to anaplasmosis. Out of 127 positive samples, four samples were customed to sequencing revealed 98-99% homology with published sequences for other available global isolates. Multi-single nucleotide polymorphisms (SNP) were observed in the sequence of two samples when aligned with the reference sequence from the NCBI database (CP023731).

Operational lessons learned in conducting an international study on pharmacovigilance in pregnancy in resource-constrained settings: The WHO Global Vaccine safety Multi-Country collaboration project.

The WHO Global Vaccine Safety Multi-Country Collaboration study on safety in pregnancy aims to estimate the minimum detectable risk for selected perinatal and neonatal outcomes and assess the applicability of standardized case definitions for study outcomes and maternal immunization in low- and middle-income countries (LMICs). This paper documents the operational lessons learned from the study. A prospective observational study was conducted across 21 hospitals in seven countries. All births occurring at sites were screened to identify select perinatal and neonatal outcomes from May 2019 to August 2020. Up to 100 cases per outcome were recruited to assess the applicability of standardized case definitions. A multi-pronged study quality assurance plan was implemented. The impact of the COVID-19 pandemic on site functioning and project implementation was also assessed. Multi-layered ethics and administrative approvals, limited clinical documentation, difficulty in identifying outcomes requiring in-hospital follow-up, and poor quality internet connectivity emerged as important barriers to study implementation. Use of electronic platforms, application of a rigorous quality assurance plan with frequent interaction between the central and site teams helped improve data quality. The COVID-19 pandemic disrupted data collection for up to 6 weeks in some sites. Our study succeeded in establishing an international hospital-based surveillance network for evaluating perinatal and neonatal outcomes using common study protocol and procedures in geographically diverse sites with differing levels of infrastructure, clinical and health-utilization practices. The enhanced surveillance capacity of participating sites shall help support future pharmacovigilance efforts for pregnancy interventions.

Estimating baseline rates of adverse perinatal and neonatal outcomes using a facility-based surveillance approach: A prospective observational study from the WHO Global Vaccine Safety Multi-Country Collaboration on safety in pregnancy.

Background: Most perinatal and neonatal deaths occur in low- and middle-income countries (LMICs), yet, quality data on burden of adverse outcomes of pregnancy is limited in such countries. Methods: A network of 21 maternity units, across seven countries, undertook surveillance for low birthweight, preterm birth, small for gestational age (SGA), stillbirths, congenital microcephaly, in-hospital neonatal deaths, and neonatal infections in a cohort of over 85,000 births from May 2019 - August 2020. For each outcome, site-specific rates per 1,000 livebirths (or per 1,000 total births for stillbirth) and 95% confidence intervals (CI) were calculated. Descriptive sensitivity analysis was conducted to gain insight regarding underreporting of four outcomes at 16 sites. Findings: Estimated rates varied across countries and sites, ranging between 43·3-329·5 and 21·4-276·6/1000 livebirths for low birthweight and preterm birth respectively and 11·8-81/1,000 livebirths for SGA. No cases of congenital microcephaly were reported by three sites while the highest estimated rate was 13/1,000 livebirths. Neonatal infection and neonatal death rates varied between 1·8-73 and 0-59·9/1000 livebirths respectively while stillbirth rates ranged between 0-57·1/1000 total births across study sites. Results from the sensitivity analysis confirmed the underreporting of congenital microcephaly and SGA in our study. Interpretation: Our study establishes site-specific baseline rates for important adverse perinatal and neonatal outcomes and addresses a critical evidence gap towards improved monitoring of benefits and risks of emerging pregnancy and neonatal interventions. Funding: The study was sponsored by the World Health Organization with funding from the Bill and Melinda Gates Foundation.

Assessment of risk factors associated with prevalence of gastrointestinal parasites in poultry of central plain zone of Punjab, India.

BACKGROUND AND AIM: Parasitic diseases are an important hurdle to the economy for the developing poultry industry due to their deleterious effects resulting into malnutrition, diminished feed conversion ratio, weight loss, decreased egg production, and mortality in young birds. The aim of this study was to assess the prevalence and associated risk factors of gastrointestinal (GIT) parasites in poultry farms of central plain zone of Punjab. MATERIALS AND METHODS: A total of 490 pooled droppings and 351 intact intestines of poultry from slaughterhouses from seven districts of central plain zone of Punjab state, India, were collected and analyzed from September 2016 to May 2018 by qualitative and quantitative techniques. RESULTS: An overall prevalence of GIT parasites was 38.36% with significantly (p<0.01) highest (74.1%) in Ludhiana and lowest (12.0%) in Shri Fatehgarh Sahib. The most predominant (86.2%) infection was coccidia. The birds reared under a deep litter system were having a higher (p<0.01) fecal load of helminthic eggs and coccidian oocysts (54.4%) compared to the cage system (37.5%). Infection rate was apparently more (40%) in broilers than layers (35.7%). Prevalence of GIT parasites was higher (p<0.01) in monsoon season (58.5%) and lower in summer season (24.48%). The broilers in the age group of 0-2 weeks possessed a significant higher (p<0.05) level of GIT parasitic infection (57.5%), while in case of layers, a higher infection rate (46.66%) was observed in birds between 9 and 18 weeks of age as compared in other groups. Higher (p<0.05) infection rate of GIT parasites was seen in crossbred (45.55%) birds as compared to desi birds (20.00%). CONCLUSION: The study showed that coccidiosis was the predominant infection among all GIT parasites based on fecal and intestinal tract content analysis. The risk factors associated with the prevalence of GIT parasitic infections were geographical location, deep litter system, broilers, age, crossbred breeds, and monsoon season.

WHO global vaccine safety multi-country collaboration project on safety in pregnancy: Assessing the level of diagnostic certainty using standardized case definitions for perinatal and neonatal outcomes and maternal immunization.

Standardized case definitions strengthen post-marketing safety surveillance of new vaccines by improving generated data, interpretation and comparability across surveillance systems. The Global Alignment of Immunization Safety Assessment in Pregnancy (GAIA) project developed standardized case definitions for 21 key obstetric and neonatal terms following the Brighton Collaboration (BC) methodology. In this prospective cohort study, we assessed the applicability of GAIA definitions for maternal immunization exposure and for low birth weight (LBW), preterm birth, small for gestational age (SGA), stillbirth, neonatal death, neonatal infection, and congenital microcephaly. We identified the missing data elements that prevented identified cases and exposures from meeting the case definition (level 1-3 of BC diagnostic certainty). Over a one-year period (2019-2020), all births occurring in 21 sites (mostly secondary and tertiary hospitals) in 6 Low Middle Income Countries and 1 High Income Country were recorded and the 7 perinatal and neonatal outcome cases were identified from routine medical records. Up to 100 cases per outcome were recruited sequentially from each site. Most cases recruited for LBW, preterm birth and neonatal death met the GAIA case definitions. Birth weight, a key parameter for all three outcomes, was routinely recorded at all sites. The definitions for SGA, stillbirth, neonatal infection (particularly meningitis and respiratory infection) and congenital microcephaly were found to be less applicable. The main barrier to obtaining higher levels of diagnostic certainty was the lack of sonographic documentation of gestational age in first or second trimester. The definition for maternal immunization exposure was applicable, however, the highest level of diagnostic certainty was only reached at two sites. Improved documentation of maternal immunization will be important for vaccine safety studies. Following the field-testing of these 8 GAIA definitions, several improvements are suggested that may lead to their easier implementation, increased standardization and hence comparison across studies.

Development and application of multiplex PCR assay for the simultaneous detection of Babesia vogeli, Ehrlichia canis and Hepatozoon canis in dogs.

A multiplex PCR assay was standardized and evaluated to simultaneously detect the DNA of Babesia vogeli, Ehrlichia canis and Hepatozoon canis in dogs of selected districts of Punjab state, India. Amplicons of 602 bp, 380 bp and 306 bp corresponding to B. vogeli (18S rRNA gene), E. canis (VirB9 gene), and H. canis (18S rRNA gene) were obtained, without any non-specific amplification. The results of multiplex PCR assay were further compared with the corresponding singleplex PCR assay. The diagnostic sensitivity and specificity of multiplex PCR assay with respect to singleplex PCR assay in the detection of B. vogeli, E. canis and H. canis varied from 50% to 100% and 92.08% to 98.79%, respectively revealing "moderate" to "very good" agreement by kappa value statistics. Blood samples from 322 dogs collected from selected districts of Punjab state, India, when screened by microscopy revealed the prevalence of B. vogeli, E. canis and H. canis as 0.31%, 0.93% and 1.86%, respectively whereas with multiplex PCR assay the values were 0.93%, 10.24% and 4.65%, respectively, with concurrent infection of E. canis & H. canis (1.86%) and B. vogeli & E. canis (0.31%). The diagnostic sensitivity and specificity of multiplex PCR assay with respect to microscopy in the detection of B. vogeli, E. canis and H. canis varied from 69.15% to 100% and 85.11% to 92.33%, respectively revealing "fair" agreement by kappa value statistics and the data was statistically significant. The analytical sensitivity of multiplex PCR assay in the detection of B. vogeli, E. canis and H. canis was 100 pg, 10 pg and 0.1 pg, respectively, whereas the values for the singleplex counterpart were 0.1 pg, 0.01 pg and 0.01 pg. Furthermore, various risk factors viz. age, breed, sex, season and districts were non-significantly associated with the prevalence of these haemoparasites except for E. canis that revealed a significant association with districts by multiplex PCR assay. Therefore the multiplex PCR assay developed may be useful in identification of the aetiological agents of these diseases during their early phase, which may in turn be useful in development of better health care and appropriate treatment of suspected dogs, particularly in endemic regions.

A Prospective Cohort Study on the Safety of Infant Pentavalent (DTwP-HBV-Hib) and Oral Polio Vaccines in Two South Indian Districts.

BACKGROUND: Safety of pentavalent (DTwP-HBV-Hib) vaccine has been a public concern in India and other countries. This study attempted to document the association of serious adverse events following immunization (AEFI, including hospitalizations and deaths of all causes) with the 3 doses of pentavalent and oral poliovirus (OPV) vaccines. METHODS: A cohort of 30,688 infants in 2 south Indian districts were enrolled and followed-up between October 2014 and May 2016, following their first vaccination with DTwP-HBV-Hib and OPV at public health facilities. During weekly follow-ups, by telephone or home visits, the serious AEFIs (hospitalizations and deaths) occurring any time after each vaccination until 4 weeks after third dose were documented. The incidence risk ratios (IRRs) of serious AEFIs in the first (days 0-6) and fourth weeks (days 21-27) after the vaccine doses were compared using the poisson regression analysis. RESULTS: Of the 30,688 infants enrolled, 30,208 received their third doses of vaccines. During the 4-week periods following each vaccination, there were 365 hospitalizations and 17 deaths. Adjusted incidence risk ratio of 3 doses combined for post-vaccination serious AEFIs during the first week compared with fourth week was 0.8 [95% confidence interval: 0.6-1.0]. CONCLUSIONS: There was no increased risk of a serious AEFIs during the first week after any of the 3 doses of pentavalent and OPV vaccination compared with the fourth week. In the absence of any temporal clustering, mortality and hospitalization rates observed in vaccinated infants probably reflects the natural occurrence of such events.

Circulating level of heat shock protein 27 is elevated in dogs with mammary tumors.

In the current study, we have synthesized canine recombinant Hsp27 in E. coli and raised hyperimmune serum against the protein in mice. Using the hyperimmune serum, an indirect ELISA was developed to estimate circulating levels of Hsp27 in dogs with various types of mammary neoplasia and to compare their levels with those of tumor-free apparently healthy dogs. The developed assay had a high specificity (83.3%) and sensitivity (96.6%) for a cut-off value of 0.45 with respect to histopathological examination in discriminating healthy dogs from those bearing mammary tumors. Serum Hsp27 levels were found to be significantly elevated in tumor subjects (20.33 ± 1.23 ng/ml) as compared to healthy controls (6.56 ± 0.51 ng/ml) and the increase in the Hsp27 levels was irrespective of mammary tumor histotypes. However, dogs with grade-II tumors had higher Hsp27 levels as compared to grade-I types. Therefore, Hsp27 can be exploited as one of the 'neoplastic signatures' of canine mammary tumors.

Development of loop-mediated isothermal amplification (LAMP) assay for detection of Hepatozoon canis infection in dogs.

The laboratory diagnosis of canine hepatozoonosis, caused by Hepatozoon canis is tedious, especially in chronic and latent infections. In the present investigation, a loop mediated isothermal amplification (LAMP) assay was developed and standardized targeting the partial 18S rRNA gene (GenBank accession no. KU096058). The LAMP primers specifically amplified H. canis DNA, whereas no amplification was detected in DNA samples from dogs infected with Babesia vogeli, B. gibsoni, Ehrlichia canis and Trypanosoma evansi, and no amplification was observed in DNA samples from H. canis-free dogs. The threshold sensitivity level of the assay was determined to be 15 fg of genomic DNA of H. canis. Furthermore, evaluation of blood samples collected from 250 dogs presented at Small Animal Clinics, Teaching Veterinary Clinical Complex, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, Punjab (India) was carried out for the presence of H. canis by microscopy, 18S PCR assay and LAMP assay. Of the total samples subjected to these tests, LAMP detected H. canis in 75 samples, while 18S PCR and microscopy detected H. canis in 28 and 9 samples, respectively. The present investigation has developed, for the first time, a highly sensitive, specific and rapid LAMP assay for the detection of H. canis, which has practical applications for the screening of field samples.

Clinical Validation of Mobile Cardiotocograph Device for Intrapartum and Antepartum Monitoring Compared to Standard Cardiotocograph: An Inter-Rater Agreement Study.

Objective: Electronic fetal monitoring (EFM) using cardiotocograph (CTG) is commonly used both to assess fetal wellbeing in late antepartum and for intervention during intrapartum period. We validated the performance of indigenously developed mobile cardiotocograph (CTG) device with wireless probes compared to standard CTG device. Materials and methods: We sequentially used mobile and standard CTG devices in 495 pregnant women in labour and 359 pregnant women with gestation > 32 weeks. The CTG interpreted by two independent obstetricians in a blinded manner were compared to estimate the agreement by kappa (k) statistic. Results: High level of agreements between mobile and standard CTG devices for both intrapartum (87.9%; kappa 0.61) and antepartum monitoring (91.2%; kappa 0.60) were observed. Most of the pregnant women (80% in intrapartum and 70% in antepartum groups) and all nurses and obstetricians preferred the mobile CTG device over standard CTG device. Conclusion: The mobile CTG device can reliably be used for both intrapartum and antepartum monitoring instead of the standard CTG devices. The smaller size, portability and ability to transmit the recordings for second opinion make it suitable for use by midwives for appropriate triaging and referral. Wider availability of CTG and interpretation support at the peripheral facilities would assist identifying at-risk pregnancies and foetuses for timely referral and appropriate action to reduce perinatal deaths, stillbirths and birth asphyxi.

Quantitative polymerase chain reaction based quantification of Brucella DNA in serum of pre- and post-therapeutic occupationally exposed infected human population.

BACKGROUND: Brucellosis is one of the neglected zoonotic diseases in humans. The serological methods based on antibody detections are unable to detect the effectiveness of treatment in humans as antibodies persist for long time in humans even after therapy. Therefore, we developed qPCR technique to overcome such discrepancy and device a rapid and efficient test for both diagnosis and follow up of the brucellosis affected individuals. METHODS: High risk suspected individuals with positive serology (RBPT, STAT and iELISA) and PCR were mainly analyzed for DNA quantification by qPCR assay. The bcsp-31 gene, a shared gene of Brucella species was amplified by genus specific primers and cloned to pGEMT™ easy vector and the cloned plasmid were used to construct a standard curve (R2=0.99, efficiency=1.98) over 7 orders of magnitude with sensitivity of ≈10 copy number. The assay was found 100% specific. RESULTS: Overall 85 individuals were found positive out of 188. Out of them, 23 serological, PCR and qPCR positive individuals were recommended for 45days therapy according to WHO regimen (Doxycycline and Rifampin) and each case was further followed by qPCR. The mean threshold cycle (Cq) before treatment was 26.05±0.347 (3940.5copies/µl), which increased significantly to 32.7±0.66 (259.13copies/µl) on 4th week during treatment, 35.12±3.12 (38.52copies/µl) at 6th week on day of treatment completion, 35.6±0.66 (34.21copies/µl) on 21st day after treatment depicting a significant reduction in DNA load over the course of treatment. Serological follow up showed that only 3 individuals had decreased STAT titre but no change in RBPT results. Out of 17 symptomatic individuals under therapy, 10 improved clinically, 5 improved clinically with persistent weakness and 2 had no effect of therapy. CONCLUSION: The study suggests that qPCR is more useful and rapid test to follow treated individuals than serology.