Prenatal exposure to ethion caused maternal and foetal toxicity in rats.

Ethion is a class II moderately toxic organothiophosphate pesticide. The main objective of this study was to evaluate the maternal and foetal toxicity of ethion in rats. Pregnant rats were divided into 5 groups. Group I served as control. Group II, III, IV, and V were orally administered with 0.86, 1.71, 3.43, and 6.9 mg/kg of ethion respectively, from gestational day (GD) 6-19. Dams were sacrificed on GD 20. Maternal toxicity was assessed by body weight gain, foetal resorptions, oxidative stress, liver and kidney function tests, and histopathology. Foetal toxicity was assessed by physical status, gross, teratological and histopathological examination. Ethion caused dose-dependent reduction in maternal body weight gain, increased resorptions, and reduced gravid uterine weights. Elevated MDA levels and altered levels of GSH, SOD and catalase were recorded in pregnant dam serum and tissues. SGOT, SGPT, total bilirubin, urea, uric acid, and creatinine were elevated in ethion groups indicating liver and kidney toxicity. Histology of uterus revealed myometrial degeneration and mucosal gland atrophy in uterus of pregnant dams and degenerative changes in placenta. It showed histological alterations in liver, kidney, and lungs. There was reduction in the foetal body weights and placental weights, and degenerative changes in the foetal liver and kidney. Gross evaluation of foetuses showed subcutaneous hematoma. Skeletal evaluation showed partial ossification of skull bones, costal separation, and agenesis of tail vertebrae, sternebrae, metacarpals and metatarsals. The findings reveal that prenatal exposure to ethion caused maternal and foetal toxicity in rats.

Enhancement of progesterone biosynthesis via kisspeptin stimulation: Upregulation of steroidogenic transcripts and phosphorylated extracellular signal-regulated kinase (p-ERK1/2) expression in the buffalo luteal cells.

The presence of Kisspeptin (Kp) and its receptors in the corpus luteum (CL) of buffalo has recently been demonstrated. In this study, we investigated the role of Kp in the modulation of progesterone (P4) synthesis in vitro. The primary culture of bubaline luteal cells (LCs) was treated with 10, 50, and 100 nM of Kp and Kp antagonist (KpA) alongside a vehicle control. The combined effect of Kp and KpA was assessed at 100 nM concentration. Intracellular response to Kp treatment in the LCs was assessed by examining transcript profiles (LHR, STAR, CYP11A1, HSD3B1, and ERK1/2) using quantitative polymerase chain reaction (qPCR). In addition, the immunolocalization of ERK1/2 and phosphorylated ERK1/2 (p-ERK1/2) in the LCs was studied using immunocytochemistry. Accumulation of P4 from the culture supernatant was determined using enzyme-linked immunosorbent assay (ELISA). The results indicated that LCs had a greater p-ERK1/2 expression in the Kp treatment groups. A significant increase in the P4 concentration was recorded at 50 nM and 100 nM Kp, while KpA did not affect the basal concentration of P4. However, the addition of KpA to the Kp-treated group at 100 nM concentration suppressed the Kp-induced P4 accumulation into a concentration similar to the control. There was significant upregulation of ERK1/2 and CYP11A1 expressions in the Kp-treated LCs at 100 nM (18.1 and 37fold, respectively, p < 0.01). However, the addition of KpA to Kp-treated LCs modulated ERK1/2, LHR, STAR, CYP11A1, and HSD3B1 at 100 nM concentration. It can be concluded that Kp at 100 nM stimulated P4 production, while the addition of KpA suppressed Kp-induced P4 production in the buffalo LCs culture. Furthermore, an increment in p-ERK1/2 expression in the LCs indicated activation of the Kp signaling pathway was associated with luteal steroidogenesis.

Outer membrane vesicles as nanovaccine candidates against pathogenic Leptospira in experimental Guinea pig model.

Leptospira interrogans serovar Hardjo is a long slender bacterium of size 0.1-0.3 µm × 5-50 µm. It is one of the major causes of bovine leptospirosis and is of economical importance because of the reproductive failure, still birth, abortion, and reduced productivity in cattle. It is also a zoonotic disease-causing infection in humans characterized by headaches, fever, chills, sweats and myalgia, lethargy, aching joints, pulmonary haemorrhages, and death in severe cases. Control of the disease involves antibiotic therapy, management and vaccination, of which immunization is the cheapest and effective means of disease prevention. The present study was developed to isolate and characterize the outer membrane vesicles of Leptospira interrogans serovar Hardjo and to evaluate their vaccine potential in guinea pig model. The OMVs were isolated from the culture by sonication and ultracentrifugation. In transmission electron microscopy, the isolated OMVs appeared as small spherical structures of 50-200 nm size. In Western blot and indirect ELISA, antibodies specific to OMVs were observed as indicative of a good humoral immune response elicited by L. interrogans serovar Hardjo OMV. The OMV-based Leptospira vaccine was able to prevent kidney lesions and renal colonization compared to the control and bacterin vaccinated group as proven by histopathology and PCR.

Experimental infection of foot and mouth disease virus (FMDV) upregulates the expression of Coxsackie and adenovirus receptor (CAR) in the myocardium of suckling mice.

The relative overexpression of Coxsackie and adenoviral receptor (CAR) predisposes children to viral myocarditis. As the foot and mouth disease virus (FMDV) causes fatal myocarditis in calves, lambs, and piglets and belongs to the same family as the Coxsackie virus, we investigated the role of CAR in FMDV induced myocarditis in the suckling mice model. Swiss albino suckling mice of 5 days (n = 24) were divided into two equal groups. One group was inoculated with suckling mice adapted FMDV serotype O at 10 LD50, while the other group served as uninfected control. In addition, adult mice (n = 12) served as the control for age related CAR expression and lack of pathogenicity to FMDV. The establishment of myocarditis was confirmed by histopathological changes typical of myocarditis along with immunolocalization of FMDV antigens in the heart of suckling mice. The FMDV inoculated suckling mice group showed a significant upregulation of CAR transcripts by 2.5 folds, overexpression of CAR protein by densitometric analysis of immunoblots, and intense immunolocalization of CAR in the sarcolemma and intercalated discs of cardiomyocytes as compared to the uninfected suckling mice group and adult mice. It was concluded that FMDV infection induced overexpression of CAR in the myocardium of suckling mice.

A retrospective study showing a high rate of seropositivity against SARS-CoV-2 in wild felines in India.

We report a high rate of seropositivity against SARS-CoV-2 in wild felines in India. Seropositivity was determined by microneutralization and plaque reduction neutralization assays in captive Asiatic lions, leopards, and Bengal tigers. The rate of seropositivity was positively correlated with that of the incidence in humans, suggesting the occurrence of large spillover events.

Pathological and molecular studies on elephant endotheliotropic herpesvirus haemorrhagic disease among captive and free-range Asian elephants in India.

In the present research pathology and molecular diagnosis of elephant endotheliotropic herpes virus-haemorrhagic disease (EEHV-HD) among Asian elephants was studied. Out of 76 cases, 20 were positive for EEHV infection in PANPOL and POL1 based semi-nested PCR. Out of 20 samples, 10 samples were fatal cases of EEHV-HD while 10 were of either subclinical or latent infection. Acute onset haemorrhagic disease with EEHV-HD had anorexia, facial and neck swelling, cyanotic buccal mucosa and tongue, nasal and ocular discharge, and colic. The hallmark of gross finding in all cases were severe haemorrhagic lesions in the internal organs viz. cyanosis of tongue with multifocal petechial haemorrhages, diffuse epicardial and endocardial haemorrhages, swollen liver (rounded edges) with parenchymal haemorrhages, serosal and mucosal haemorrhages in gastrointestinal tract, congested kidneys with corticomedullary haemorrhages, highly congested meninges, and brain capillaries with haemorrhages. Microscopic findings in all the cases had severe vascular changes in the visceral organs. Microthrombi was present in the vasculature of tongue, heart, lung, liver, kidney, and brain. The endothelial lining of most of the blood vessels were swollen with apoptotic changes. Amphophilic to basophilic intranuclear inclusion bodies were observed in the endothelial cells. Immunostaining using anti-EEHV DNAPOL hyperimmune sera revealed intense positive signals in the endothelium of blood vessels and their walls. Quantification of viral load in necropsy tissue samples revealed highest in the heart (7.4 × 106/µg of sample) and least in the brain (9 × 103/µg of sample). The PCR amplicons from EEHV1 specific genes (POL1(U38) and TER were subjected to partial genome sequencing which had 99.9% similarity with the EEHV1A subtype. It was concluded that Asian elephants in India are latently infected for EEHV1 and in all the fatal EEHV-HD cases, EEHV1A subtype was the causative agent with characteristic pathomorphological changes in visceral organs.

Differential expression of inflammatory cytokines, prostaglandin synthases and secretory leukocyte protease inhibitor in the endometrium and circulation in different graded CEH-pyometra in bitch.

Cystic endometrial hyperplasia (CEH)-pyometra (CEH-P) is one of the most common reproductive disorders in bitches, posing a risk to both future fertility and life. The aims of the current study were to elucidate the differential expression patterns of inflammatory mediators at transcript and protein levels in the endometrium and to assess the concentrations of key inflammatory mediators in the peripheral circulation of bitches with different graded CEH-P. A total of 25 client-owned intact mixed breed bitches of 3-10 years presented to the outpatient department of RVP-TVCC of the institute were considered for the study. Of which, 22 cases suggestive of pyometra and 3 cases of CEH obtained during routine elective ovariohysterectomy were subjected to histopathological examination. Uteri were categorized into CEH (n = 3), moderate CEH-P (mCEH-P, n = 9), severe CEH-P (sCEH-P, n = 6) and atrophic pyometra (AT-P, n = 7). A group of age matched (n = 12) bitches without pyometra served as control. Endometrial transcripts such as IL6, IL8, PTGS2, PGFS, and SLPI were expressed differentially in the CEH and CEH-P bitch. In addition, a strong immunoreactivity (IR) of IL6, IL8, PTGS2, and mPGES1 was recorded in the sCEH-P uterus, while expression of IL10 was noticed in AT-P. In circulation, serum IL6 was the most relevant marker with high sensitivity of 96.2% and specificity of 84.6% at a cut off concentration 8.5 pg/mL followed by SLPI with 95.2% sensitivity, and 84.6% specificity at cut off concentration of 1.3 ng/mL. Serum IL10, PGFM and SLPI concentration in the peripheral circulation were 1.5-2.23 fold higher in mCEH-P, 0.87-2.5 fold higher in sCEH-P and 2.9-3.5 fold higher in AT-P than that of control. It is concluded that monitoring the serum concentration of IL6, IL10 and SLPI would be useful adjunct to the established hematobiochemical parameters in the management of pyometra in the bitch with critical illness.

Genetic and population diversity of Toxocara cati (Schrank, 1788) Brumpt, 1927, on the basis of the internal transcribed spacer (ITS) region.

The present investigation was aimed to study the sequence, phylogenetic and haplotype analyses of Toxocara cati based on the ITS region, along with the genetic diversity, demographic history and population-genetic structure. The maximum likelihood tree based on Kimura 2-parameter model was constructed using the complete ITS region of all the nucleotide sequences (n = 57) of Toxocara spp. and other related ascarid worms available in the GenBank™. It placed all the sequences of T. cati into four major clades designated as T. cati genotypes 1-4 (TcG1-G4). A total of 66 signature nucleotides were identified in the ITS region between genotypes. The median-joining haplotype network displayed a total of 24 haplotypes, with China exhibiting the highest number of haplotypes (h = 20) followed by India (h = 4), and Japan and Russia (h = 1). It indicated a clear distinction between all the four genotypes. The pairwise FST values between all the genotypes indicated huge genetic differentiation (> 0.25) between different T. cati genotypes. Moreover, the gene flow (Nm) between T. cati genotypes was very low. Results of AMOVA revealed higher genetic variation between genotypes (92.82%) as compared to the variation within genotypes (7.18%). The neutrality indices and mismatch distributions for the G1-G4 genotypes, Indian isolates and the overall dataset of T. cati indicated either a constant population size or a slight population increase. The geographical distribution of all the genotypes of T. cati is also reported. This is the first report of genotyping of T. cati on the basis of the ITS region.

An evidence of Humanin-like peptide and Humanin mediated cryosurvival of spermatozoa in buffalo bulls.

Buffalo spermatozoa are vulnerable to cryo-injuries due to inherent deficiency of endogenous antioxidants, high polyunsaturated fatty acids (PUFA) content in plasma membrane and low cholesterol/phospholipid (C/P) ratio. Humanin is a potent cytoprotective agent that protects the cells against oxidative stress and apoptosis. The present study was designed to establish the presence of Humanin in buffalo and effect of Humanin supplementation on freezability of buffalo spermatozoa. Indirect immunofluorescence test revealed presence of Humanin in ejaculated and epididymal spermatozoa, and, elongated spermatids and interstitial space in the testicular tissue section. Humanin levels in seminal plasma were significantly and positively correlated with sperm concentration and individual progressive motility (IPM) in good (n = 22; IPM >70%) and poor (n = 10; IPM <50%) quality ejaculates. For supplementation studies, a total of 24 ejaculates (IPM ≥70%) were collected and each ejaculate was then divided into four aliquots. First aliquot was diluted with egg yolk-tris-glycerol (EYTG) extender without Humanin and served as control group (Group I). Rest three aliquots were diluted with extender containing 2 (Group II), 5 (Group III) and 10 µM Humanin (Group IV), respectively. Semen was cryopreserved using standard protocol and evaluated at pre-freeze for lipid peroxidation (LPO) and post-thaw stages for spermatozoa kinematics, LPO, mitochondrial membrane potential (MMP), capacitation, apoptotic status and DNA integrity. The treatment group that showed best results (5 µM) was compared with control group for in vitro fertility assessment by homologous zona binding assay. The LPO levels were lower (p < 0.05) in 5 and 10 µM Humanin supplemented group. The MMP and DNA integrity were higher (p < 0.05) in 5 µM group than other groups. F-pattern was higher (p < 0.05) and B-pattern was lower (p < 0.05) in 5 and 10 µM Humanin supplemented groups. Lower apoptotic and higher viable spermatozoa (p < 0.05) were observed in 5 µM Humanin group. The mean number of spermatozoa bound to zona pellucida was higher (p < 0.05) in 5 µM Humanin treated group than the control group. The study established the presence of Humanin in buffalo spermatozoa and seminal plasma for very first time and concluded that Humanin supplementation at 5 µM concentration improves the freezability and in vitro fertility of buffalo spermatozoa.

Tuberculosis in free-ranging and captive wild animals: Pathological and molecular diagnosis with histomorphological differentiation of granulomatous lesions.

Tuberculosis (TB) is a serious zoonotic threat, impacting the human-livestock-wildlife interface globally. Here, we evaluated the status and histomorphological differentiation of TB lesions in 89 morbid cases of wild animals (bovids, cervids, carnivores, non-human primates, and pachyderms) in India. Histomorphological and molecular studies were done using Ziehl-Neelsen (ZN) staining, immunohistochemistry, and polymerase chain reaction (PCR), whereas cultural isolation was performed on selected samples. A total of 32 (35.95%) cases were confirmed as TB, comprising of 12 carnivores, 09 bovids, 06 cervids, 04 non-human primates, and a pachyderm. The TB lesions in the lungs, liver, and lymph nodes varied from the large-sized caseous nodules filled with dry cheesy material in bovids and cervids to variable-sized cavitations containing liquefied caseum in carnivores' lungs. The lungs, livers, and spleens of non-human primates exhibited small to medium-sized nodules. Histologically, lesions were divided into four categories (Types I, II, III, and IV) based on the extent of necrosis, the presence of mineralization, giant cells, and fibrous encapsulation. Extensive caseous necrosis with calcification, abundant giant cells, and thick fibroblastic encapsulation were consistent findings in the lungs, livers, and lymph nodes of bovids and cervids, whereas airway impaction with cellular exudate containing a teeming number of acid-fast bacilli and, at times, alveolar rupture leading to cavity formation was present in the lungs of carnivores. Absence of calcification and fibrous encapsulation was recorded in lungs of non-human primates. Immunohistochemical labelling with anti-early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) antibodies showed mild, moderate, and intense positive reactions in type II and III, type I, and type IV granulomatous lesions, respectively. Molecular detection by PCR revealed Mycobacterium tuberculosis (12 carnivores, 02 non-human primates and 01 pachyderm), M. bovis (02 cervids and 01 bovid) and M. orygis (02 cervids and 01 bovid). Cultural isolation confirmed M. tuberculosis in 03 carnivores and M. orygis in 02 cervids and 01 bovid. Our findings imply that TB is quite prevalent in the wildlife of India and there are considerable differences in the histomorphological lesions induced by distinct Mycobacterium species in different wild animals. The circulation of TB organisms in wild animals warrants a strict surveillance programme to identify the carrier status of these animals so that effective TB control strategies can be formulated to prevent spillover and spillback incidences at the human-livestock-wildlife interface.

First report on the detection of Japanese encephalitis virus in fruit bats from India.

Japanese encephalitis (JE) is a mosquito borne viral zoonotic disease and JE virus (JEV) is responsible for causing several children deaths every year in India. Since 1978, cases of JE have been reported from Gorakhpur district of Uttar Pradesh state annually. The knowledge on the role played by wildlife reservoirs in the sylvatic transmission and maintenance of JE virus remains limited. Bats are reservoir hosts for several emerging and re-emerging viral pathogens but their role in zoonotic cycle of JEV has not been elucidated yet. In Gorakhpur district of Uttar Pradesh, 52 fruit bats were found dead on 26 May 2020. The post-mortem report of the bat samples conducted at the Indian Veterinary Research Institute stated that the bats died due to brain hemorrhage, caused by excessive heat. The brain tissue samples of the bats were subjected to investigation using molecular techniques to determine the presence of JEV. The present work reports for the first time the detection of JEV in brain samples of bats from India. The viral load ranging from 8 to 18 copies/reaction was detected in brain samples by TaqMan real Time RT-PCR. The low viral load might be the reason for the absence of apparent clinical signs in bats and suggests the probable role of fruit bats in maintaining the JEV in nature.

Molecular and pathological screening of canine distemper virus in Asiatic lions, tigers, leopards, snow leopards, clouded leopards, leopard cats, jungle cats, civet cats, fishing cat, and jaguar of different states, India.

The present investigation was conducted to rule out canine distemper (CD) diseases in Indian wild felids (Asiatic lions, tigers, leopards, snow leopards, clouded leopards, leopard cats, jungle cats, civet cats, fishing cat, and jaguar). The collected samples were screened for CD virus (CDV) by histopathology (HP), immunohistochemistry (IHC) and reverse transcriptase-polymerase chain reaction (RT-PCR) targeting H gene and N gene. The HP and IHC of suspected samples portrayed that 22 [11 leopards, 6 lions, 3 tigers, 1 snow leopard and 1 civet cat] out of 129 (17.05%) wild felids were positive for CD. The major pathological consequences were observed in spleen, lung, kidney and brain. The syncytia and intranuclear as well as intracytoplasmic eosinophilic inclusion bodies were seen in CDV infected cells. Although the histopathological lesions in spleen were more specific and consistent, however, the severe demyelinated leukoencephalitis (usually expected in CD infected dog) was not observed in the brain of any Indian wild felids. Conversely, the CDV antigen has been portrayed via IHC in pancreatic islets of Langerhans of tiger species for the first time in this study. Moreover, the concurrent CD and babesiosis has also been observed in a lioness without a usual coffee-coloured urine. The N gene and H gene of CDV isolates were amplified, sequenced and subsequently constructed the phylogenetic tree. The phylogenetic analysis of H gene revealed that the CDV isolates from Indian lion formed separate clade with CDV isolates from Indian dog and Indian palm civet cat. Furthermore, two CDV isolates from Indian tigers formed clade with Onderstepoort vaccine strain and CDV isolates from dogs of Uttar Pradesh, USA and UK. Evidently, CDV is circulating in Indian wild felids and causing diseases in them.

Natural infection of Delta mutant of SARS-CoV-2 in Asiatic lions of India.

The current pandemic caused by a novel coronavirus (SARS-CoV-2) has underlined the importance of emerging diseases of zoonotic importance. Along with human beings, several species of wild and pet animals have been demonstrated to be infected by SARS-CoV-2, both naturally and experimentally. In addition, with constant emergence of new variants, the species susceptibility might further change which warrants intensified screening efforts. India is a vast and second most populated country, with a habitat of a very diverse range of animal species. In this study we place on record of SARS-CoV-2 infections in three captive Asiatic lions. Detailed genomic characterization revealed involvement of Delta mutant (Pango lineage B.1.617.2) of SARS-CoV-2 at two different locations. Interestingly, no other feline species enclosed in the zoo/park were found infected. The epidemiological and molecular analysis will contribute to the understanding of the emerging mutants of SARS-CoV-2 in wild and domestic animals.

Expression of kisspeptin and its receptor in different functional classes of ovarian follicle in the buffalo (Bubalus bubalis).

Recently, we reported the differential expression of kisspeptinergic system in the bubaline hypothalamus and corpus luteum. Here, we document the expression of kisspeptin (Kp) and its receptor (Kiss1r) in the ovarian follicles of the buffalo with respect to the functional status. Follicles of ≥10 to ≤13 mm diameter (n = 45) were retrospectively categorized into active (n = 18), intermediate (n = 16) and atretic (n = 11) follicles based on the concentrations of intrafollicular progesterone (P4) and estradiol (E2). The P4:E2 ratio was significantly lower in the active follicle (0.43 ± 0.08) than that of the intermediate (3.46 ± 0.53) and atretic (28.4 ± 10.6) follicles (P < 0.05). Relative fold change in the transcripts of kisspeptin (Kiss1), Kiss1r, gonadotrophin receptors, steroid acute regulatory protein (StAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), cytochrome P450 Family 19 subfamily A member 1 (CYP19A1), insulin like growth factor -1 (IGF-1), apoptotic factors (caspase 3 and B-cell lymphoma 2, BCL2) was calculated using qPCR in the follicular wall of the three categories of follicle (n = 8/group). In another experiment, histological sections of the ovary (n = 41) were used to group the follicles as described above and immunostaining of Kp, Kiss1r and aromatase was done. A significant upregulation of StAR, CYP11A1 and CYP19A1 in the active follicles supported the endocrine basis of follicular classification. The transcripts of Kiss1 and Kiss1r were upregulated by 19.45 fold and 4.25 fold, respectively in the active follicle as compared to other groups. Immunolocalization studies revealed that Kp and Kiss1r were localized to the basal and antral granulosa cells (GC) of the active and intermediate follicles; however, the staining intensity was stronger in the former group. Strong expression of CYP19A1 in the GC layer of active follicle supported the histological basis of defining the functional status of the follicle. It is concluded that the follicular compartment of the bubaline ovary expressed the constituents of kisspeptinergic system. The expression of Kp and Kiss1r was influenced by the functional status of the follicle with intense localization in the GC layer of the active follicles.

A study on clinical diagnosis of tuberculosis in free ranging and captive wild animals of India.

Background: Tuberculosis (TB) is a disease of paramount importance at the wildlife-livestock-human interface. Aims: To study the occurrence and Mycobacterium (M) species involved in the TB of free-ranging and captive wild animals in various Indian states. Methods: A total of 396 clinical samples from 207 different wild animal species from various Indian national parks, zoological gardens, etc., were analyzed by lateral flow assay (LFA), Ziehl-Neelsen (ZN) staining, and PCR. Clinical samples include blood (n=156), faecal swabs (n=103), serum (n=73), and nasal swabs or trunk wash fluids (n=64). Results: Clinical signs of TB were absent in 202 animals, although 21 wild animals were seropositive for pathogenic Mycobacterium antigens by LFA. Clinical signs like progressive weight loss, and respiratory distress were exhibited by 4 sloth bears (Melursus ursinus) and an elephant (Elephas maximus), which were also found positive for LFA, PCR, and ZN staining. ZN staining showed positivity for acid-fast bacilli (AFB) in 9 (8.74%) faecal and 9 (14.06%) nasal swabs or trunk wash fluids of sloth bears (7 samples) and elephants (2 samples). M. tuberculosis was detected in 7 sloth bears and 2 elephants, whereas M. bovis was found in a spotted deer (Axis axis) by species-specific PCR. Conclusion: The circulation of TB organisms in wild animals warrants a strict surveillance programme to identify the carrier status of these animals so that effective TB control strategies can be formulated.

Sparganosis mimicking a soft-tissue tumor: A diagnostic challenge.

Human sparganosis is a rare but important food borne zoonosis and could be attributed to increased consumption of raw meat of fish, frogs, snakes etc. Sparganosis may involve varied organ systems but subcutaneous sparganosis remains the one of the commonly reported clinical condition. Rarity of this problem reinforces the necessity of sensitising the treating physicians of the differential possibility of this infection in patients with history of practice of consuming raw meat. Expansion of health communication and provision of safe food and water by the civic agencies can be a part of powerful preventive strategies.

Isolation and genetic characterization of canine adenovirus type 2 from a domestic dog showing neurological symptoms.

Canine adenoviruses (CAVs) are of two types: canine adenovirus type 1 (CAV-1), which causes infectious canine hepatitis, and canine adenovirus type 2 (CAV-2), which is mainly associated with the respiratory type of disease in dogs. Due to the widespread use of modified live vaccines to control canine adenoviral infections and subsequently reduced disease incidence, CAVs are often neglected by clinicians. Although a number of studies are available about CAV-1 prevalence in India, only meagre information is available about CAV-2. This study reports the CAV-2 infection in a vaccinated dog with neurological and respiratory symptoms which was found negative for other canine pathogens like canine distemper virus and canine parvovirus. The virus was successfully isolated from rectal swab in MDCK cells and characterized by immunofluorescence assay and virus neutralization test. On phylogenetic analysis of partial E3 region, the Indian CAV-2 grouped in a separate clade different from established subgroups. An insertion of "G" nucleotide was reported at nucleotide (nt.) position 1077 in the E3 gene of Indian CAV-2 isolates which led to a frameshift in the coding region of E3 gene thereby imparting additional eleven amino acids to its C-terminal end in comparison to isolates from other parts of the world. This may have an implication on the functional role of E3 protein inside the cell. This study reinforces the unique signature insertion in the E3 gene of Indian CAV-2 and is the second study in the world to report the association of CAV-2 with neurological disease in dogs.

Multi-drug resistant (MDR), extended spectrum beta-lactamase (ESBL) producing and carbapenem resistant Escherichia coli in rescued Sloth bears (Melursus ursinus), India.

The study reports the multi-drug resistant (MDR), extended spectrum beta-lactamase (ESBL) producing and carbapenem resistant Escherichia coli (CRE) isolated from rescued sloth bear (Melursus ursinus), India. Non-duplicate faecal samples from 21 adult rescued sloth bears were collected at once during 2015-2016 and processed for isolation of E. coli and antibacterial susceptibility pattern. From 21 samples, 45 E. coli were isolated and on phenotypic screening, 23 were MDR, 17 were ESBL producers, and five were carbapenem-resistant (CR). Three E. coli isolates (6.67%, 3/45) showed no resistance, however 42 isolates (93.33%, 42/45) exhibited resistant to at least one antibiotics. The MDR isolates carried beta-lactamase, chloramphenicol, aminoglycosides, tetracycline, fluroquinolone, and sulphadimidine resistance genes. All the phenotypic ESBL producing isolates harbored blaCTX-M genes. On genotypic screening, three CRE (60.0%, 3/5) were positive for blaNDM carbapenemase gene and efflux pump-mediated carbapenem resistance was detected in two CRE isolates (40.0%, 2/5) which were negative for carbapenemase genes. The CRE isolates (n = 5) also co-harbored AMR genes like blaTEM-1, blaAmpC, qnrA, qnrB, qnrS, tetA, tetB and sulI. Virulence screening of the resistant isolates detected the presence of Stx1(n = 1), Stx2 (n = 3), eaeA (n = 4) and hlyA (n = 3) genes. Plasmid incompatibility (Inc) typing revealed that two isolates harboured blaNDM-5 gene on Incl1 and one isolate on IncF plasmid. Apart from the NDM gene, the plasmids also carried tetracycline, beta-lactamase and quinolone resistance genes. The plasmid multilocus sequence typing (pMLST) of the E. coli Incl1 plasmid showed the Sequence Type (ST) 297. This appears to be the first report of MDR, ESBL producing and blaNDM-5 genes on Incl1 and IncF plasmids from rescued sloth bear.

Genetic characterization and phylogenetic analysis of Sarcocystis suihominis infecting domestic pigs (Sus scrofa) in India.

A total of 57 tissue samples of domestic pigs (Sus scrofa) were collected from the meat outlets of five north Indian states and examined for sarcocystosis by histological and molecular methods. The genomic DNA extracted from five representative positive isolates was subjected to PCR amplification of the partial 18S rRNA gene followed by cloning and sequencing. Sequence analysis of the newly generated Indian isolates recorded 96.9-100.0% identity with published sequences of Sarcocystis suihominis. Two new haplotypes that have not been previously described manifested 99.5-100.0% nucleotide homology within themselves. In the phylogenetic analysis, Indian isolates of S. suihominis grouped together with S. suihominis originating from Italy, and they collectively formed a sister clade with Sarcocystis miescheriana within a clade containing various Sarcocystis spp. of ruminants having felids as final hosts. At the same time, this clade separated from a sister clade containing Sarcocystis spp. of bovid or cervid ruminants using canids as known or surmised definitive host. The current study established the phylogenetic relationship of Indian isolates of S. suihominis with various Sarcocystis spp. as well as with other taxa of Sarcocystidae family based on 18S rRNA gene for the first time.

Expression of Kisspeptin and its receptor in the hypothalamus of cyclic and acyclic buffalo (Bubalus bubalis).

Kisspeptin (Kiss1), neurokinin-B (NKB) and dynorphin (Dyn) neurons regulate the surge and pulsatile centres of gonadotropin releasing hormone (GnRH) in the hypothalamus and are modulated by the ovarian steroids. Accordingly, we studied the temporospatial expression of Kiss1, its receptor and other genes that regulate GnRH in the preoptic area (POA) and arcuate (ARC) regions of hypothalamus at different phases of bubaline estrous cycle. Brain of buffalo (n = 32) was collected immediately after exsanguination and categorized into early luteal (EL), mid luteal (ML), follicular (FL) stages and acyclic (n = 8/group). Total RNA was extracted from the POA and ARC of each stage and real time PCR amplification of Kiss1, Kiss1r, NKB, NKB receptor (NKBR), Dyn, Dyn receptor (OPRK1), GnRH1, ERα, PR, LEPR and GHSR was done using GAPDH as endogenous control and acyclic stage as calibrator group. Further, immunolocalization of Kiss1 and Kiss1r was done on the hypothalamus. In the POA, significant up-regulation of Kiss1 and NKB with a concomitant down-regulation of Dyn transcripts was recorded at FL stage. There was, however, down-regulation of Kiss1 and Kiss1r during the EL perhaps due to the loss of estradiol as a consequence of ovulation. On the other hand, in the ARC, there was a significant up-regulation of Kiss1 and Dyn at FL and ML, while NKB transcript was consistently down-regulated at any stage of estrous cycle. In the POA, expression of ERα was not modulated; however, PR was down-regulated in the EL. In the ARC, the ERα expression was significantly up-regulated in the EL, whereas, PR was moderately expressed irrespective of the stage of estrous cycle. The immunolocalization study revealed the presence of Kiss1 and Kiss1r in the POA and ARC in the cyclic buffalo with relative abundance at FL. The transcriptional profile of the genes suggests that there is estrous cycle stage specific expression of Kiss1, Kiss1r and other GnRH regulating genes in the POA and ARC regions of hypothalamus in the buffalo. Up-regulation of Kiss1r in the POA during ML and ARC during EL indicates the involvement of kisspeptinergic system in the regulation of low LH pulse frequencies during the early and mid luteal phases in the cyclic buffalo.