Bovine cerebral theileriosis: first molecular report in cross bred cattle calf in India.

Bovine tropical theileriosis caused by Theileria annulata, is a serious constraint to Indian dairy industry with more fatal infections in exotic cattle and substantial losses to cross-bred and indigenous zebu cattle. The present communication is to place on record the first report of molecular based confirmed case of cerebral theileriosis caused by T. annulata coupled with its morphological detection, clinical manifestations, haematological alterations and therapeutic management in a cross bred cattle calf from India. After preparation of peripheral thin blood smear from cross bred cattle calf at the site of collection and fixation with methanol, blood sample brought to Department of Veterinary Parasitology, College of Veterinary Science and A.H, Jabalpur and stained by standard protocol for Giemsa staining. Genomic DNA was isolated from the collected blood sample using QIAamp® DNA blood mini kit following the manufacturer's recommendations and PCR was performed. The cross bred cow calf revealed high rise in temperature (105.5°F), increased heart rate, labored breathing with seromucous nasal discharge, enlargement of prescapular lymph node and animal exhibited tonic clonic convulsions in response to any sudden noise. Giemsa stained thin blood smear revealed intraerythrocytic piroplasm and Koch'sblue bodies of T. annulata within the cytoplasm of lymphocytes. The species of Theileria was confirmed by molecular amplification of genomic DNA as T. annulata.

Molecular identification of Theileria species in naturally infected sheep using nested PCR-RFLP.

Ovine theileriosis is an important tick-borne haemoprotozoan disease of sheep in tropical and subtropical regions, causing severe productivity and economic loss. There is a paucity of information related to molecular studies of ovine theileriosis from India. The present study identified different Theileria spp. in naturally infected sheep using nested PCR-restriction fragment length polymorphism (nPCR-RFLP). Blood samples and ticks were collected from 204 sheep in different agro-climatic zones of Haryana state, India, during the tick active season. Microscopic examination of thin blood smears revealed 33.3% (68/204) infections with Theileria spp., while 44.6% (91/204) of blood samples were positive by nPCR assay. Different Theileria spp. were identified based upon RFLP patterns using four restriction enzymes: Hpa II, Bsh 1285I, Hae II and Rsa I. Out of 91 positive samples, 50.5% (46/91), 23.08% (21/91), 11% (10/91) and 2.2% (2/91) were positive for T. ovis, T. lestoquardi, T. luwenshuni (Theileria sp. China 1/Theileria sp. China) and T. annulata, respectively. Mixed infection was detected in 13.2% (12/91) of cases. Based upon HpaII enzymatic digestion pattern, two samples with T. lestoquardi and T. annulata, nine samples with T. lestoquardi and T. ovis and one sample with T. ovis and T. annulata were detected. The presence of these Theileria spp. was further confirmed by sequence analysis. The majority of ticks collected from sheep were identified as Rhipicephalus spp. followed by Hyalomma anatolicum and Hemaphysalis spp. The present investigation depicts the first comprehensive molecular report of naturally infected sheep with T. ovis, T. lestoquardi, T. annulata and T. luwenshuni from northern India.

First report of molecular and phylogenetic analysis of Physaloptera praeputialis in naturally infected stray cats from India.

Nematodes of the genus Physaloptera are globally distributed and infect a multitude of hosts. Their life cycle involves orthopterans and coleopterans as intermediate hosts. The morphological characters alone are inadequate to detect and differentiate Physaloptera spp. from its congeners. Moreover, molecular studies are limited to compare them precisely. The present communication reports the first molecular phylogenetic characterization of feline Physaloptera spp. from India based on mitochondrial cytochrome c oxidase subunit 1 (COX1) and small subunit ribosomal DNA (18S rDNA). The nematodes were first isolated from the stomach of adult stray cats during necropsy examination. Based on the gross and microscopic characters, the worms were identified as P. praeputialis. Morphological identification was further confirmed through PCR targeting the barcode region of the mitochondrial cytochrome c oxidase subunit I (MT-COI) gene, using nematode-specific primers cocktail followed by species specific primers targeting partial COX1 and 18S rRNA genes. Generated sequences were submitted in NCBI GenBank (MW517846, MW410927, MW411349), and phylogenetic trees were constructed using the maximum likelihood method. When compared with other sequences of Physaloptera species across the globe, the present isolates showed 85.6-97.7% and 97.3-99% nucleotide homology based on COX1 and 18S rRNA gene, respectively. BLASTn analysis revealed a strong identity to other Physaloptera spp., and the phylogenetic tree placed all Physaloptera spp. in the same cluster. This study again indicates the usefulness of molecular techniques to substantiate the identity of species that may lack adequate descriptions and impart new insight for the potentially overlooked significance of P. praeputialis infections in felines.

First report of the isolation and phylogenetic characterization of equine Setaria digitata from India based on mitochondrial COI, 12S rDNA, and nuclear ITS2 sequence data.

Equine ocular setariasis arising mainly from ectopic infestation of Setaria digitata is a common vision impairing ophthalmic disease in India, and the identification of this filarial nematode is based solely on morphology. However, morphological characters alone are inadequate to detect and differentiate S. digitata from its congeners. The present communication reports the first phylogenetic characterization of equine S. digitata from India based on sequences derived from the mitochondrial cytochrome c oxidase subunit 1 (COI), the mitochondrial small subunit ribosomal DNA (12S rDNA), and the nuclear internal transcribed spacer 2 (ITS2). Three isolates were characterized for each gene, and respective sequences were submitted to NCBI database (MN078131, MN078132, and MN095798). The sequences were also compared with the other related sequences available from PubMed around the globe, and phylogenetic analysis was carried out in conjunction with nucleotide homologies. There was no intraspecific variation among the Indian isolates. The phylogenetic analysis of S. digitata, inferred from these genes, showed that the isolate sequences obtained from different host species created a separate monophyletic clade within the genus Setaria with minor sequence variations revealing similar molecular characteristics of S. digitata isolates throughout the globe. In addition, the studied Indian isolates were found closer to Sri Lankan isolates. The S. digitata and S. labiatopapillosa appeared as sister species.

Targeted transcript analysis revealed association of suboptimal expression of certain endometrial immunity-related genes with disparate uterine diseases in zebu cows.

The onset of uterine infection during postpartum period compromises uterine health, fertility, and productivity of dairy cattle. Endometrial innate immunity plays a key role in eliminating uterine infection and keeping the uterus healthy. Hence, the present study has been designed with the hypothesis that altered endometrial immune response around calving may compromise uterine health during postpartum period. Expression of interleukins (IL-1ß, IL-6, IL-8, IL-10, and TNF-α), prostaglandin synthase (PGFS, PGES), and antimicrobial peptides (beta-defensins (BDEF-4, BDEF-5), lingual antimicrobial peptide (LAP), and calcium-binding proteins (S100A8, S100A9, and S100A12) in endometrial tissues on the day of calving was studied using qRT-PCR, and circulating concentrations of prostaglandin E and F metabolites (PGEM and PGFM) during peripartum period (on days - 7, - 4, - 1 (before calving), 0 (on the day of calving), + 1, + 4, and + 7 (post calving)) of normal (healthy) cows (n = 11) that did not develop postpartum uterine infection and cows that developed puerperal metritis (n = 7) and clinical endometritis (n = 6) were studied. Endometrial expression of IL-1ß, TNF-α, BDEF-4, BDEF-5, S100A8, S100A12, and PGFS was higher (P < 0.05), and expression of IL-6, IL-8, IL-10, and PGES was lower (P < 0.05) in normal (healthy) cows than puerperal metritic and clinical endometritic cows. The PGFM concentration in serum was high (P < 0.05) on days 0, + 1, and + 4 of calving in puerperal metritic cows followed by normal and clinical endometritic cows. However, PGEM concentration in serum was high (P < 0.05) during peripartum period in uterine-infected (puerperal metritic and clinical endometritic) cows compared with normal cows. From the above findings, it is concluded that higher constitutive expression of IL-1ß, TNF-α, PGFS, BDEF-4, BDEF-5, S100A8, and S100A12 genes in the endometrium and lower concentration of PGEM during the period immediate to calving might be beneficial for uterine health of cows.

Global proteomic analysis of water buffalo (Bubalus bubalis) saliva at different stages of estrous cycle using high throughput mass spectrometry.

Accurate and efficient detection of estrus is one of the major constraints for exploitation of the production potential of buffalo owing to its poor manifestation of estrus signs, seasonal differences in expression and higher incidences of silent estrus (29%). The current study focused on identification of estrus specific candidate proteins in saliva of buffaloes. Estrus was detected based on behavioral signs in response to the teaser and changes in reproductive organs and confirmed by per-rectal examination, trans-rectal USG of reproductive organs, cervico-vaginal mucus characteristics and blood serum progesterone estimation. Day of onset of estrus was considered as day 0 and day -3, +3, +10 were considered as proestrus, metestrus and diestrus stage of the estrous cycle respectively. A total of 19 animals and their 38 estrous cycles (two from each) were included in this study. Saliva was collected from these animals during different stages of estrous cycle. Out of these, 08 animals were selected for global proteome analysis of saliva using in-solution digestion and nano-LC-MS/MS. A total of 275, 371, 304 and 565 proteins were identified with ≥2 peptides during proestrus, estrus, metestrus and diestrus stages of estrous cycle. Among the identified proteins 31, 62, 32 and 104 proteins were found specific to proestrus, estrus, metestrus and diestrus stage of the estrous cycle. Few salivary proteins such as Cullin-associated NEDD8-dissociated protein 1, Heat shock 70 kDa protein 1A, 17-beta-hydroxysteroid dehydrogenase type 1, Inhibin beta A chain, testin were identified as estrus specific and are important for estrus physiology. Taken together, these estrus specific proteins could be considered as the candidate biomarker for detection and confirmation of estrus in buffalo after thorough validation.

Pseudomalaria in a domestic pigeon: a case report.

Microscopic examination of thin blood smear of a domestic pigeon brought to Teaching Veterinary Clinical Complex, Junagadh, Gujarat revealed mature and immature halter shaped gametocytes of Haemoproteus columbae encircling the nucleated erythrocytes. The disease was diagnosed as acute infection of pigeon malaria more notably known as pseudomalaria. The parasitic infection was further confirmed by polymerase chain reaction assay targeting the Cytochrome b (cyt b) gene fragment amplifying 207 base pair (bp) fragment. Pseudolynchia canariensis, the vector for H. columbae was also recovered from underneath the feathers of the affected pigeon.

Prevalence of ovine cystic echinococcosis in Kashmir Valley, North India.

A total of 1455 local and non-local (originating from other Indian states), slaughtered or spontaneously dead, sheep in various areas of Kashmir Valley were investigated for the presence of cystic echinococcosis over a period of one year. The overall prevalence was 7.97% with higher prevalence in local (14.3%) than in non-local sheep (6.06%). The prevalence of infection, total number of cysts recovered and mean intensity of infection were higher in lungs as 66.2%, 506 & 5.1% respectively, followed by liver (28.5%, 169, 3.9%) and spleen (5.3%, 9, 1.13%). Either single (71.55%) or multiple (28.45%) organ involvements were observed. 66.6% of cysts were of small size, 19.29% medium, 7.01% large and 7.01% calcified. The fertility of cysts was noted to be 65.7% whereas 34.2% were infertile which included 27.1% sterile and 7.01% calcified cysts. The viability percentage of protoscolices from all the fertile cysts was 74.2%. The number of cysts recovered was higher in sheep with body condition score- emaciated, thin and average, and lower in, fat and obesed. The study showed that the local sheep were more vulnerable to contract cystic echinococcosis than non-local sheep which is further aggravated by poor body condition.

Transcriptional abundance of antioxidant enzymes in endometrium and their circulating levels in Zebu cows with and without uterine infection.

Oxidative stress during peripartum period may compromise the uterine immunity. In the present study, we assessed the oxidative stress and antioxidant status during peripartum period and studied their relationship with postpartum uterine infection in dairy cows. Peripheral blood concentrations of total antioxidant capacity (TAC), malondialdehyde (MDA) and nitric oxide (NO) were determined (day -21, -7, on the day of calving and day +7, +21, +35) in normal (n=11), puerperal metritic (n=7) and clinical endometritic (n=6) cows. Endometrial biopsy was performed on the day of calving and expression of CAT, GPx4 and SOD2 genes was studied using qRT-PCR. Puerperal metritic cows had significantly (P<0.05) lower TAC (on day -7, day 0, day +7, +21 & +35), higher MDA (on day -21, -7 & on the day of calving) and NO (on day 0, +7 & day +35) concentrations compared to normal cows. Similarly, clinical endometritic cows had significantly (P<0.05) lower TAC (on day -7, 0, +7 & +21), higher MDA (on day -21, -7, +7 and +35) and NO (on day +7, +21 & +35) concentrations compared to normal cows. The expression of CAT and GPx4 genes was lower (P<0.05) and SOD2 gene was higher (P<0.05) in endometrial tissue of cows that developed uterine infection compared to normal cows. The relationship of peripheral levels of MDA and NO with antioxidant enzymes expression in endometrial tissue was found significant. Receiver operator characteristic analysis revealed that the concentrations of TAC on day -7 to day +35, MDA on day -21 to day +7 and NO on the day of calving to day +35 were highly correlated to the development of postpartum uterine infection in cows. It may be inferred that the low serum TAC level and high level of lipid peroxidation and NO during peripartum period influenced the endometrial expression of anitioxidative genes that compromised the uterine health during postpartum period.

Prevalence of gastrointestinal parasites in bovines in and around Junagadh (Gujarat).

Coprological examination of 416 bovine faecal samples revealed the presence of parasitic stages of Toxocara vitulorum, strongyles, Strongyloides spp., Fasciola spp., amphistomes, coccidia (Eimeria spp. and Cryptosporidium spp.) and Buxtonella sulcata. About 42 % (n = 302) faecal samples from cattle and 36 % (n = 114) samples from buffaloes were positive for gastrointestinal (GI) parasitic infections. Both cattle (14.57 %) and buffalo (15.79 %) had the highest incidence of Buxtonella sulcata, respectively. The overall incidence of GI parasitic infections in young animals (below 1 year) was higher followed by older (more than 5 years) and adult animals (1-5 years) and the difference was statistically significant (p < 0.05). Non descriptive breeds of bovines showed more parasitic infections than pure breeds, the difference being statistically non-significant (p > 0.05). Season wise GI parasitic infections were recorded to be non-significantly (p > 0.05) higher in monsoon (48.38 %) followed by summer (39 %) and winter (34.61 %) in cattle. There was no significant variation of GI infections in buffaloes in relation to season though highest prevalence was documented in monsoon (44.89 %) followed by winter (35.71 %) and summer (24.32 %). Similarly, sex wise females recorded higher infection rates than males in bovines and the difference being statistically non-significant (p > 0.05).

Seasonal incidence of parasitic diseases in bovines of south western Gujarat (Junagadh), India.

The current study was conducted to investigate the incidence of parasitic diseases in bovines which were sick and brought at veterinary hospital for treatment. A total of 366 samples were investigated from cattle (n = 175) and buffaloes (n = 191) presented at Teaching Veterinary Clinical Complex (TVCC), Veterinary College, Junagadh, Gujarat during January to December 2014. Examination of Giemsa-stained peripheral blood smears exhibited that 58.6 % of cattle and 41.2 % of buffaloes were infected with haemoparasites comprising Babesia bigemina, Theileria annulata, and Anaplasma marginale @ of 54.0, 3.4 and 1.1 in cattle and 38.8, 1.2 and 1.2 percent in buffaloes, respectively. The incidence of total haemoparasites and B. bigemina infections was significantly higher (p < 0.05) in cattle whereas, incidence of haemoparasites were recorded significantly higher in the month of July to November (i.e., rainy and autumn) in both cattle and buffaloes, respectively (p < 0.01 and p < 0.001). Coprological examination revealed that the overall incidence of gastrointestinal (GI) parasitic infection was 45.5 % in cattle and 43.4 % in buffaloes. The incidence of individual parasite was 11.4, 1.1, 2.3, 4.5, 1.1, 3.4, 2.3 and 19.3 in cattle and 4.7, 0.9, 0.0, 2.8, 0.9, 5.7, 0.0 and 28.3 % in buffaloes for Eimeria spp., Trichuris spp., Toxocara vitulorum, Strongyle, Fasciola spp., amphistomes, Schistosoma indicum and Buxtonella sulcata, respectively which differ insignificantly (p > 0.05). Seasonal prevalence of GI parasites was highest in summer in both cattle and buffaloes, the data being statistically non-significant (p > 0.05). However, the incidence of B. sulcata in both cattle (19.3 %) and buffaloes (28.3 %) was higher in comparisons to other GI parasites. The present investigation emphasized that B. bigemina and B. sulcata are the most important parasites of bovines of this region.

An overview on kinetoplastid paraflagellar rod.

Kinetoplastids, the evolutionary ancient organisms exhibit a rich and diverse biology which epitomizes many of the fascinating topics of recent interest and study. These organisms possess a multifunctional organelle, the flagellum containing a canonical 9 + 2 axoneme which is involved in vital roles, viz. parasite cell division, morphogenesis, motility and immune evasion. Since Antony Van Leeuwenhoek's innovative explanation of 'little legs' helping the movements of microbes in 1975, this biological nanomachine has captured the thoughts of scientists. The core structure of kinetoplastid flagellum is embroidered with a range of extra-axonemal structures such as paraflagellar rod (PFR), a large lattice like structure which extends alongside the axoneme from the flagellar pocket to the flagellar tip. The coding sequences for significant components of PFR are highly conserved throughout the Kinetoplastida and Euglenida. The high order organization and restricted evolutionary distribution of the PFR components and structure makes the PFR a particularly valuable therapeutic and prophylactic target. This review focuses on the recent developments in identification of ultra structural components of PFR in order to understand the function of this intriguing organelle and devising strategies for therapeutic interventions.

Association of milk components with intra-mammary inflammation in Jaffrabadi buffaloes.

AIM: To study the alteration of major milk components such as milk fat, protein, lactose, solid not fat (SNF) and total solids (TS) and their association with different degree of intra-mammary inflammation (IMI) in Jaffrabadi buffaloes. MATERIALS AND METHODS: Milk samples (n=1516) were collected from Jaffrabadi buffaloes separately from each quarter. Milk samples were analyzed for milk fat, protein, lactose, SNF and TS percent on the same day using milk analyzer "LACTOSCAN." Milk samples were checked for IMI by California mastitis test (CMT), and the results were expressed as negative (0), +, ++, and +++ CMT score. The traits of milk components which showed significant difference (p<0.05) between samples from inflamed and non-inflamed quarters were analyzed by receiver operating characteristic (ROC) analysis to see the accuracy and degree of association with IMI. RESULTS: Among several milk components, milk protein and lactose percent showed a significant difference (p<0.05) between milk samples from normal and inflamed quarters. Though, during the early stage of mammary gland inflammation milk protein percent remained significantly high (p<0.05), later with an increase in the degree of severity of inflammation it did not show any difference. Milk samples from normal udder quarters had significantly higher lactose percent than inflamed quarters (p<0.05). Milk lactose percent decreased gradually with an increase in the degree of severity of inflammation. ROC analysis revealed that milk samples having lactose content below the threshold values had significantly higher chances to come from inflamed udder quarters (p<0.05). Though, the value of the area under curve (AUC) indicated that milk lactose was significantly associated with IMI (p<0.05), the accuracy was moderate (AUC=0.71-0.75). CONCLUSIONS: The results of the present study indicated that milk lactose percent gradually and significantly reduced during IMI and can be used as a marker for identification of IMI in buffaloes. However, ROC analysis further confirmed that using milk lactose IMI can be identified with moderate accuracy.

Development of cathepsin-L cysteine proteinase based Dot-enzyme-linked immunosorbent assay for the diagnosis of Fasciola gigantica infection in buffaloes.

Native cathepsin-L cysteine proteinase (28 kDa) was purified from the excretory secretory products of Fasciola gigantica and was used for sero-diagnosis of F. gigantica infection in buffaloes by Dot-enzyme-linked immunosorbent assay (Dot-ELISA). The test detected F. gigantica field infection in these animals with a sensitivity of ∼ 90%. No specific IgG antibody binding was displayed by sera obtained from 76 buffaloes considered to be Fasciola and other parasite-free by microscopic examination of faeces and necropsy examination of liver, rumen and intestine. Additionally, sera from 156 Fasciola-free buffaloes, yet infected with Gigantocotyle explanatum, Paramphistomum epiclitum, Gastrothylax spp., Strongyloides papillosus and hydatid cyst were all negative, indicating that F. gigantica cathepsin-L cysteine proteinase does not cross-react with these helminth parasites in natural infection of the host. The data indicated that cathepsin-L cysteine proteinase based Dot-ELISA reached ∼ 90% sensitivity and 100% specificity with relation to above parasites in the detection of bubaline fasciolosis. The present Dot-ELISA diagnostic assay is relevant to the field diagnosis of F. gigantica infection in buffaloes.

Lack of protective efficacy in buffaloes vaccinated with Fasciola gigantica leucine aminopeptidase and peroxiredoxin recombinant proteins.

Gene coding for leucine aminopeptidase (LAP), a metalloprotease, was identified in the tropical liver fluke, Fasciola gigantica; that on sequence analysis showed a close homology (98.6%) with leucine aminopeptidase of the temperate liver fluke, Fasciola hepatica. The recombinant leucine aminopeptidase protein was expressed in Escherichia coli. F. gigantica peroxiredoxin, a hydrogen peroxide scavenger and an immunomodulating protein, was also cloned and expressed in E. coli. A vaccination trial in buffaloes was conducted with these two recombinant proteins, with 150 and 300 µg of leucine aminopeptidase and a cocktail of 150 µg each of recombinant leucine aminopeptidase and peroxiredoxin in three groups, respectively. Both Th1- and Th2-associated humoral immune responses were elicited to immunization with these antigens. A challenge study with 400 metacercariae did not show a significant protection in terms of reduction in the worm burden (8.4%) or anti-fecundity/embryonation effect in the immunized groups, as to the non-immunized control animals. Our observations in this buffalo vaccination trial are contrary to the earlier promise shown by leucine aminopeptidase of F. hepatica as a leading candidate vaccine molecule. Identification of leucine aminopeptidase gene and evaluation of the protein for its protective efficacy in buffaloes is the first scientific report on this protein in F. gigantica.

Control of poultry coccidiosis: changing trends.

Coccidiosis is the most important protozoan disease affecting the poultry industry worldwide. Control of poultry coccidiosis is presently based on managerial skills and the use of prophylactic coccidiostatic drugs. With the emergence of drug resistant Eimeria strains, emphasis has been laid on development and use of safer vaccines; some of them have been commercialized successfully. The present review deals with the various factors responsible for the development of clinical coccidiosis in poultry as well as an overview of the currently available inducers and boosters of immunity against coccidiosis. There are three groups of vaccines currently available against coccidiosis which can be distinguished on the basis of characteristics of the Eimeria species included in the respective products, viz. vaccines based on live virulent strains, vaccines based on live attenuated strains, and vaccines based on live strains that are relatively tolerant to the ionophore compounds. The latter vaccine combines the early chemotherapeutic effect of ionophores with the late prophylactic effect of vaccination. Although in the near future more varieties of oocyst based live vaccines are expected, identification of selective coccidian-specific immunoprotective molecules is likely to get more attention to facilitate the sustainable control of poultry coccidiosis.