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.

Comparative epidemiology and pathophysiology of patent and latent babesiosis caused by Babesia bigemina in buffaloes and cattle from different agroclimatic zones of Punjab State, India.

To conduct comparative epidemiology of parasitologically positive (patent) and polymerase chain reaction positive (latent) cases of bovine babesiosis in Bet Region (low-lying areas adjoining Sutlej, Beas, Ravi, and Ghaggar rivers of Punjab) of diverse agroclimatic zones of Punjab state in relation to haematobiochemical parameters as patho-physiological markers, blood samples from 783 dairy animals (487 buffaloes and 296 cattle) were analysed parasitologically by Giemsa-stained blood smears (GSBS) and by molecular-based polymerase chain reaction (PCR) targeting SpeI-AvaI restriction fragment of Babesia bigemina. We ruled out the endemicity of the disease with 2.17% patent and 3.96% latent cases of B. bigemina with significantly higher prevalence (P < 0.01) in cattle than buffaloes. The spatial distribution for a guideline to local veterinary practitioners and policy-makers indicated highest number of patent and latent cases in western zone and undulating plain zone, respectively. District wise highest prevalence of patent as well as latent infection observed in SBS Nagar of undulating plain zone showed substantial agreement (Kappa value: 0.70) between the two techniques. Haematology revealed marked microcytic hyperchromic anaemia in patent animals of group I (GSBS positive; n = 17) and latent animals of group II (PCR positive; n = 14) as compared to disease-free controls (group III; n = 10). Blood urea nitrogen (BUN), creatinine and gamma-glutamyltransferase (GGT) levels were significantly increased (P < 0.05) in group I in comparison to group II and group III indicated comparative pathogenic effect of babesiosis in patent cases. Though patent cases showed higher pathogenicity of babesiosis, diagnosis of latent infection is significant as it may act as source of infection for spread to other highly prone bovines.

First record of Toxoplasma gondii antibodiesin Royal Bengal tigers (Panthera tigris tigris) and Asiatic lions (Panthera leo persica) in India.

The purpose of this study was to detect the antibodies against Toxoplasma gondii in Royal Bengal tigers (Panthera tigris tigris), Asiatic lions (Panthera leo persica), leopards (Panthera pardus), and elephants (Elephas maximus indicus) residing in the Mahendra Chaudhury Zoological Park, in Chhatbir, Punjab (India) during winter and monsoon seasons. Using  indirect ELISA, 20 serum samples were analysed during the winter season. Results indicated that 1 lion (5%) tested seropositive, and 3 tigers and 1 lion (20%) were considered suspect. During the monsoon, 4 individuals (2 tigers and 2 lions, 20%) were seropositive, whereas only 1 tiger (5%) gave suspected results. Significantly higher globulin, creatinine, blood urea nitrogen, phosphorus, and creatine kinase values were recorded in seropositive and suspected groups. Levels of albumin, glucose, calcium, sodium, and iron decreased significantly in the seronegative group. Results from sero-testing 40 rodents trapped in and around the park depicted the presence of antibodies against Toxoplasma gondii in 1 individual. This study reveals the haemato-biochemical alterations in both seropositive and suspected wild felids for toxoplasmosis. Moreover, it provides the first serological evidence of T. gondii exposure in wild felids, notably Royal Bengal tigers and Asiatic lions, in India.

Evaluation and Correlation of Multiple Anthelmintic Resistances to Gastrointestinal Nematodes Using Different Fecal Egg Count Reduction Methods in Small Ruminants of Punjab, India.

PURPOSE: Concordance of multiple anthelmintic resistances for gastrointestinal nematodes in small ruminants by three average-based and two individually based fecal egg count reduction (FECR) tests was evaluated and corrected. METHODS: Sheep and goats (≥ 8 weeks) from five farms were randomly assigned to three treatment groups (I, II, III; n = 10 per group) and one untreated control group (Group IV; n = 10). Group I received fenbendazole at the dose rate of 5 and 10 mg/kg, Group II received ivermectin at the dose rate of 0.2 and 0.4 mg/kg, and Group III received levamisole at the dose rate of 8 and 12 mg/kg body weight orally for sheep and goat, respectively. Three average-based methods of FECR (FECR1, FECR2 and FECR3) and two individually based methods of FECR (iFECR1 and iFECR2) were evaluated. RESULTS: For fenbendazole resistance, Spearman correlation coefficient for FECR1 was non-significant with other formulae, but for FECR2 with FECR3, FECR3 with iFECR1 and iFECR1 with iFECR2 coincidence was significant at 1%, while for FECR2 with iFECR2 and FECR3 with iFECR2 it was significant at 5%. Spearman correlation coefficients for ivermectin resistance were significant at 1% level and for levamisole it showed significant coincidence at 1% for FECR1 with FECR2 and iFECR1, FECR2 with FECR3 and iFECR1, and iFECR1 with iFECR2, while for FECR1 with FECR3 and iFECR2 coincidence was significant at 5% level. Concordance of kappa values indicated that the coincidence of the prevalence of anthelmintic resistance (95% CI) among the five farms was non-significant. CONCLUSIONS: Concordance between the standard average-based FECR and individually based methods suggests that either method could be applied to small ruminant farms.

Morphometric and scanning electron microscopy based identification of Ancylostoma caninum parasites in dog.

Ancylostoma caninum, a blood feeding nematode parasite (Family: Ancylostomatidae, Superfamily: Ancylostomatoidea) can cause anaemia, dark reddish-brown to black haemorrhagic diarrhoea, dehydration, wasting and deaths due to heavy blood loss. Adult hook worm parasites recovered from the intestine of a stray dog at the time of necropsy were identified as A. caninum based on morphological characters and morphometric observations involving scanning electron microscopy (SEM). Different developmental stages of hookworm eggs viz. 8 cell stage, morula, gastrula and vermiform were observed during the culture process of faecal sample. High quality SEM photographs showed teeth of dimensions 52.5, 42.3 and 23.5 µm on one side and 55.4, 43.8 and 21.0 µm on the other side along with the presence of characteristic transverse cuticular striations on body surface of A. caninum parasites.

Molecular and pathological studies on natural cases of bovine theileriosis.

The present communication is a part of study conducted on 32 cases of bovine lymphadenopathies. Out of which, six cases of bovine theileriosis were diagnosed on the basis of peripheral blood smear examination and gross lesions in lymph nodes and visceral organs. Nested PCR using two primer sets N516/N517 and 14136/294 was conducted on samples collected from affected lymph nodes confirms Theileria annulata infection in five out of six cases. Sequencing analysis of amplified product showed 92, 94 and 93 % homology of isolate TH1_Bovine, TH2_Bovine and THEN_Bovine for T. annulata with T. annulata Tamilnadu and Pant Nagar. An upregulation of Th2 cytokines in the cases of theileriosis was observed as the level of TNF-α in individual animals varies from higher value (1028 pg/100 µg protein) to as low as 500 pg/100 µg protein. An increase in level of Th2 cytokines IL-4 and IL-10 was also observed. The present study concluded that pathological studies and cytokine analysis of lymph nodes are of paramount importance in disease diagnosis and associated immune response of the animal with lymphadenopathies.

Diagnosis and management of bovine babesiosis outbreaks in cattle in Punjab state.

AIM: The aim of the present study was to diagnose severe outbreaks of bovine babesiosis in Punjab state, in the year 2015 and to suggest control and preventive measures to animal owners. MATERIALS AND METHODS: Mortality of animals was recorded in two cattle herd comprising a total of 465 cattle in Sangrur (n=125) and Faridkot (n=340) districts. There was a history of purchase of animals at one farm. 23 blood samples were collected from diseased (n=15) and healthy animals (n=8) for hematological analysis, parasitological, and polymerase chain reaction (PCR)-based diagnosis. Ticks were also collected from animals for identification. RESULTS: Out of 465 cattle at risk, 28 were critically ill and 14 died of disease with morbidity, mortality, and case fatality rate of 6.02%, 3.01%, and 50.00%, respectively. Clinical signs and necropsy findings were suggestive of babesiosis. Ticks collected from both the outbreaks were identified as Rhipicephalus (Boophilus) microplus. Thin blood smears from infected animals (especially with clinical sign of hemoglobinuria) were found positive for Babesia bigemina organisms; however, molecular diagnosis (PCR) further confirmed the disease. Animals were successfully treated with diminazene aceturate, hematinics, and antipyretics. CONCLUSIONS: Two fatal outbreaks of babesiosis in cattle were diagnosed with application of conventional parasitological, hematological, and molecular diagnostic techniques. PCR was found to be far more sensitive in detecting the disease, especially in latent infections. Animal owners were advised to follow quarantine measures before mixing new animals in the herd and strategic acaricidal treatments for effective tick control.

Molecular epidemiology, risk factors and hematochemical alterations induced by Theileria annulata in bovines of Punjab (India).

Bovine tropical theileriosis, caused by Theileria annulata, is one of the economically important fatal tick borne haemoprotozoan diseases of dairy animals. The aim of present investigation was to map the distribution of T. annulata in bovines of Punjab state of India in relation to various risk factors including age, sex of animals, location and management of farms. In a cross sectional study, a total of 1278 blood samples were randomly collected from twenty districts falling in five major agro-climatic zones of Punjab. All the samples were screened by blood smear examination followed by polymerase chain reaction targeting SSU rRNA gene for Theileria spp. PCR positive samples (n = 386) for Theileria spp. were then analyzed for T. annulata by amplification of Tams1 gene. Overall prevalence of T. annulata was found to be 29.26% in Punjab, with highest in western Zone (40.49%, 95% CI = 35.57-45.41) and lowest in submountain zone (18.90%, 95% CI = 13.73-24.06). The propensity of incidence of T. annulata was found to be highest in cross bred cattle (32.40%, 95% CI = 29.87-34.94), followed by indigenous cattle (19.64%, 95% CI = 10.67-28.61) and buffaloes (19.2%, 95% CI = 14.99-23.41). Between the two sexes, incidence of T. annulata was higher in female animals. Calves less than 6 months of age were found to be more prone to theileriosis.

Detection and assessment of risk factors associated with natural concurrent infection of Trypanosoma evansi and Anaplasma marginale in dairy animals by duplex PCR in eastern Punjab.

Duplex PCR consisting of two primer sets within a single mixture for the simultaneous detection of Anaplasma marginale and Trypanosoma evansi was standardized and employed on 219 blood samples collected from cattle (165) and buffaloes (54) from eastern Punjab to evaluate the status of concurrent infection and associated risk factors. The reaction produced 257- and 407-bp amplification products targeting repetitive nucleotide sequence of T. evansi and msp1ß gene of A. marginale, respectively. The nucleotide sequence analysis of individual amplicons expressed the fidelity of the primer pairs used; duplex PCR was 100% sensitive and 92.66 % specific with conventional microscopy for the detection of mixed infections. Among the agro-climatic zones of interest, undulating zone was at higher risk of T. evansi infection (odds ratio (OR) = 1.75, 95% confidence interval (CI) = 0.94-3.27), and submountain zone (OR = 1.89, 95% CI = 1.11-3.33) for A. marginale. For the concurrent infection, the relative risk among the two zones was almost unity. The cross-bred cattle population was at the highest risk of infection, may it be solo infection of T. evansi (OR = ∞, 95% CI = 1.18-∞)/A. marginale (OR = 6.39, 95% CI = 1.14-125.3) or dual infection (OR = ∞, 95% CI = 0.39-∞) of both as the indigenous cattle are resistant to the infection. Cross-bred cattle were at approximately three times the risk than buffaloes. For the dual infection, the cattle calves were at about 2.5 times higher risk than buffalo calves. Results indicate the endemic status of these infections in the region and mark out the commodities at great risk and requiring better surveillance.

Prevalence and morphological characterisation of Echinococcus granulosus from north India.

The purpose of this study was to document the prevalence and to analyze morphological characteristics from hydatid cysts to test their suitability for strain identification. In the present study, 4,130 animals, including 278 cattle, 298 buffaloes, 760 sheep, 2,439 goat and 355 pigs were examined for the presence of hydatid cysts on post-mortem inspection at different slaughter houses/shops in northern India. Morphological characteristics from hydatid cysts were analyzed to test their suitability for strain identification. For statistical analysis, five variables were considered: number of hooks per rostellum, blade length of large and small hooks, and total length of large and small hooks. Principal component analysis was applied for analysis of morphological parameters. Out of a total of 4,130 animals examined, 66 were positive for hydatid cysts (prevalence 1.598 %). The prevalence of hydatid cysts was highest in cattle (5.39 %) followed by buffaloes (4.36 %), pigs (3.09 %), sheep (2.23 %) and goat (.41 %). The results indicate significant prevalence of hydatidosis in all the food producing animals and further that morphological analysis can also be used as a valid criterion for differentiation of different strains of E. granulosus particularly in developing countries where molecular studies could not be performed due to lack of infrastructure or financial constraints.

Prevalence and haemato-biochemical profile of Anaplasma marginale infection in dairy animals of Punjab (India).

OBJECTIVE: To do the systematic comparison of prevalence of anaplasmosis by PCR and Giemsa stained thin blood smear (GSTBS) based parasitological assays in dairy cattle of Punjab, which has not been reported yet. To analyse the haematobiochemical alterations in infected animals to arrive at the conclusion regarding the pathogenicity induced by Anaplasma marginale (A. marginale) in latent and patent infection. METHODS: Study was conducted on 320 animals (236 cows, 62 calves and 22 buffaloes) of Punjab, India. PCR on genome of A. marginale was performed by targeting msp1 ß gene using specific primers BAP-2/AL34S, amplifies products of size 407 bp. Questionnaires based data on the characteristics of the infected animals and management strategies of the farm were collected and correlated. RESULTS: Higher prevalence and more significant association was observed in the PCR based molecular diagnosis (P=0.00012) as compared to that in GSTBS (P=0.028 8) based diagnosis with various regions under study. With respect to the regions, highest prevalence was recorded in Ferozepur by PCR based diagnosis, while that in Jalandhar by GSTBS examination. Similar marked significant association of the PCR based diagnosis with the age of the animals under study (P=0.00013) was observed elucidating no inverse age resistance to A. marginale in cow calves. Haematobiochemical profile of infected animals revealed marked anemia, liver dysfunction and increase globulin concentrate indicating rise in immunoglobulin level to counteract infection. CONCLUSIONS: PCR is far more sensitive in detecting the disease even in latent infection which may act as nidus for spread of anaplasmosis to susceptible animals in endemic areas. Severity of anaemia and liver dysfunction were comparable both in patent as well as latent infection indicating pathogenicity of both.

Molecular epidemiology of Echinococcosis from food producing animals in north India.

Echinococcosis is an important medical, veterinary and economic concern in India. Ten cysts were randomly selected from each intermediate host species (cattle, buffalo, sheep, goat and pigs). Either the germinal layer (sterile cysts) or protoscoleces (fertile cysts) were collected for molecular characterization. A 434 base pair fragment of the mitochondrial cytochrome oxidase-1 gene was amplified using PCR from each isolate. Ten representative samples (2 from each intermediate host species) were sequenced in both the directions from which readable sequences were obtained from nine for phylogenetic analysis (NCBI, Blast). Phylogenetic analysis of cytochrome oxidase I gene revealed that seven (77.7%) isolates, from cattle (2), pigs (2), buffaloes (1) and goat (2) were clustered with the Indian Buffalo (G3) strain of Echinococcus granulosus, while two (22.2%) isolates from sheep were clustered with the sheep strain (G1) of E. granulosus. Phylogenetic analysis of the cytochrome oxidase-1 gene revealed that the buffalo strain (G3) and common sheep strain (G1) are cycling among livestock in north India and that these strains are highly adapted to cattle, buffalo, sheep, goats and pigs.

Pathological studies on experimental Trypanosoma evansi infection in Swiss albino mice.

The pathology of Trypanosoma evansi infection was studied in Swiss albino mice using cattle isolate of the parasite. Sixteen Swiss albino mice were used in the experiment and were divided into two groups viz. infected group (I) and uninfected healthy control group (II) comprising 12 and four mice, respectively. Twelve mice from group I were infected with 1 × 10(5) purified trypanosomes. Systematic necropsy examination specifically of the infected mice (group I) as well as of healthy control (group II) was performed and pathological changes were recorded. The different tissue samples were collected in 10 % neutral buffered formal saline and were used to study the histopathological changes. Gross post-mortem examination revealed enlargement of spleen, petechial haemorrhages in liver in the terminal stages of disease. Tissue sections revealed presence of numerous trypanosomes in blood vessels of liver, spleen, brain and kidneys. Microscopically, liver revealed lesions varying from vacuolar degeneration, coagulative necrosis along with congestion and haemorrhages. Spleen showed extensive haemorrhages in red pulp area, haemosiderosis and aggregation of histiocytes resulting in multinuclear giant cell formation. Lungs revealed oedema, congestion and mild inflammatory changes. Brain revealed mild degenerative changes along with congestion of meningeal blood vessels. Kidneys showed tubular degeneration, congestion and cellular infiltration. Heart revealed mild degenerative changes along with interstitial oedema. All changes were consistent with trypanosome infection and were confirmed by presence of trypanosomes in most of the tissue sections examined.