Comparative Comprehensive Analysis on Natural Infections of Hymenolepis Diminuta and Hymenolepis Nana in Commensal Rodents.

This first comprehensive report from Punjab province of India relates to patho-physiological alterations alongwith morpho-molecular characterisation and risk assessment of natural infections of Hymenolepis diminuta and Hymenolepis nana in 291commensal rodents including house rat, Rattus rattus (n=201) and lesser bandicoot rat, Bandicota bangalensis (n=90). Small intestine of 53.61 and 64.95 % rats was found infected with H. diminuta and H. nana, respectively with a concurrent infection rate of 50.86 %. There was no association between male and female rats and H. diminuta and H. nana infections (ᵡ2 = 0.016 and 0.08, respectively, d.f.= 1, P>0.05), while the host age had significant effect on prevalence of H. diminuta and H. nana (ᵡ2 = 28.12 and 7.18, respectively, d.f.= 1, P≤0.05) infection. Examination of faecal samples and intestinal contents revealed globular shaped eggs of H. diminuta without polar filaments (76.50 ± 3.01µm x 67.62 ± 2.42 µm), while smaller sized oval eggs of H. nana were with 4 - 8 polar filaments (47.87 ± 1.95 µm x 36.12 ± 3.05 µm). Cestode infection caused enteritis, sloughing of intestinal mucosa, necrosis of villi and inflammatory reaction with infiltration of mononuclear cells in the mucosa and submucosa. Morphometric identification of the adult cestodes recovered from the intestinal lumen was confirmed by molecular characterisation based on nuclear ITS-2 loci which showed a single band of 269 bp and 242 bp for H. diminuta and H. nana, respectively. Pairwise alignment of the ITS-2 regions showed 99.46 % similarity with sequences of H. diminuta from USA and 100 % similarity with sequences of H. nana from Slovakia, Kosice.

Atypical theileriosis with cutaneous involvement in a cow in India: a case report.

Bovine tropical theileriosis caused by Theileria annulata is an overwhelming haemoprotozoan tick-borne disease in taurine and cross-bred cattle in Punjab, India. However, there seems to be no report from India of cutaneous nodules associated with the disease. This report describes a five-year-old cross-bred cow presented to a university clinic with a history of fever, inappetence and malaise for the past six to seven days. Clinical examination revealed normal vital parameters, pale mucous membranes, mild enlargement of the prescapular lymph nodes and multiple subcutaneous nodular masses (2-4 cm) on the neck and abdomen. Haematology revealed mild anaemia and leucopenia with 48% neutrophils, 48% lymphocytes and 4% eosinophils. Romanowsky-stained smears of fineneedle aspiration biopsy samples from swollen lymph nodes and subcutaneous masses showed an increased number of lymphoid cells, suggesting cutaneous lymphomatosis. However, a critical examination of the smears from subcutaneous nodules showed a large number of Koch's blue bodies in macrophages and lymphoblasts, and several piroplasms were also noticed within the red blood cells in lymph node smears. A peripheral blood smear revealed mild to moderate parasitaemia. Extracted DNA from the parasitologically positive blood sample was subjected to nested polymerase chain reaction (nPCR) using T. annulata speciesspecific primers encoding the 30-kiloDalton major sporozoite surface antigen. The desired 572-base pair amplified product of the nPCR was comparable to the positive control. This seems to be a rare case of T. annulata in an adult cross-bred cow, showing cutaneous nodular involvement.

La theilériose bovine tropicale est une maladie causée par le protozoaire Theileria annulata et transmise par les tiques, affectant massivement les populations de bovins et de bovidés métis au Pendjab (Inde). Il semble toutefois que la présence de nodules cutanés associés à la maladie n'y ait jamais été rapportée jusqu'à présent. Les auteurs décrivent le cas soumis à une clinique vétérinaire universitaire d'une vache métisse âgée de cinq ans qui présentait depuis six à sept jours un tableau fébrile accompagné d'une perte d'appétit et d'un affaiblissement général. À l'examen clinique, les paramètres vitaux étaient normaux mais une pâleur des membranes muqueuses a été observée, ainsi qu'un gonflement modéré des ganglions lymphatiques préscapulaires et de nombreuses masses nodulaires sous-cutanées (de 2 à 4 cm d'épaisseur) au niveau du cou et de l'abdomen. L'hématologie a mis en évidence une anémie modérée et une leucopénie, les leucocytes se répartissant en 48 % de neutrophiles, 48 % de lymphocytes et 4 % d'éosinophiles. Les frottis à coloration de Romanowsky d'une biopsie par aspiration à l'aiguille fine des ganglions lymphatiques enflés et des masses sous-cutanées ont fait apparaître une augmentation du nombre de cellules lymphatiques évocatrice d'une lymphomatose cutanée. Néanmoins, un examen critique des prélèvements de nodules sous-cutanés a permis de constater la présence d'un grand nombre de corps bleus de Koch dans les macrophages et les lymphoblastes ; en outre, de nombreux piroplasmes ont été trouvés dans les globules rouges des frottis de ganglions lymphatiques. Un frottis de sang périphérique a permis de quantifier la parasitémie comme étant de niveau faible à modéré. L'ADN extrait de l'échantillon de sang à parasitologie positive a été soumis à une amplification en chaîne par polymérase nichée (nPCR) utilisant des amorces spécifiques de T. annulata codant pour l'antigène majeur de surface (30 kDa) du sporozoïte. Le produit amplifié par nPCR de la séquence souhaitée de 572 paires de bases était similaire à celui de l'échantillon de contrôle positif. Il s'agit probablement d'un cas rare d'infection à T. annulata chez une vache adulte métisse présentant des manifestations nodulaires cutanées.

La teileriosis tropical bovina causada por Theileria annulata es una devastadora enfermedad hemoprotozoaria transmitida por garrapatas que afecta al ganado taurino e híbrido del Punjab (India). Ahora bien, en la India no parece haber ningún caso descrito de esta enfermedad que se acompañe de la presencia de nódulos cutáneos. Los autores describen el caso de una vaca de cinco años híbrida que fue presentado a una clínica universitaria con un cuadro de fiebre, pérdida de apetito y decaimiento en los seis a siete días anteriores. El examen clínico puso de manifiesto parámetros vitales normales, mucosas pálidas, leve hipertrofia de los ganglios linfáticos prescapulares y múltiples bultos subcutáneos de tipo nodular (2 a 4 cm) en cuello y abdomen. El análisis hematológico reveló una leve anemia y leucocitopenia, con un 48% de neutrófilos, un 48% de linfocitos y un 4% de eosinófilos. Tras proceder a una biopsia de ganglios inflamados y bultos subcutáneos por aspiración con aguja fina, el examen de frotis de estas muestras con tinción de Romanowsky reveló un número excesivo de células linfáticas, lo que parece apuntar a una linfomatosis cutánea. No obstante, al examinar más a fondo los frotis de nódulos subcutáneos se observó que macrófagos y linfoblastos albergaban un gran número de cuerpos azules de Koch. También se observaron varios piroplasmas dentro de los eritrocitos presentes en los frotis de ganglios linfáticos. Un frotis de sangre periférica reveló una parasitemia entre leve y moderada. El ADN extraído de esta muestra de sangre positiva fue sometido a una técnica de reacción en cadena de la polimerasa (PCR) anidada en la que se emplearon cebadores específicos de la especie T. annulata que codifican el antígeno de superficie principal de 30 kDa del esporozoíto. La deseada secuencia de 572 pares de bases amplificada por PCR resultó comparable con la correspondiente secuencia de la muestra positiva de control. Parece tratarse pues de un caso raro de infestación por T. annulata de una vaca adulta híbrida que se acompaña de nódulos cutáneos.

Periurban outbreaks of bovine calf scours in Northern India caused by Cryptosporidium in association with other enteropathogens.

Bovine calf scours reported to be caused by multiple aetiologies resulting in heavy mortality in unweaned calves and huge economic loss to the dairy farmers. Among these, cryptosporidiosis is an emerging waterborne zoonoses and one of the important causes of neonatal calf diarrhoea. Poor immune response coupled with primary cryptosporidial infections predispose neonatal calves to multiple secondary infections resulting in their deaths. In the present study, faecal samples from 100 diarrhoeic calves randomly picked up out of 17 outbreaks of bovine calf diarrhoea in periurban Ludhiana, Punjab in Northern India were subjected to conventional (microscopy, modified Zeihl-Neelsen (mZN) staining) and immunological and molecular techniques (faecal antigen capture ELISA and PCR) for detection of primary Cryptosporidium parvum infection as well as other frequently reported concurrent pathogens, viz. rotavirus and coronavirus, Salmonella spp., Escherichia coli, Clostridium perfringens and Eimeria spp. The faecal antigen capture ELISA and PCR revealed 35% prevalence of C. parvum in contrast to 25% by mZN staining with a relatively higher prevalence (66·7%) in younger (8-14-day-old) calves. The detection rate of the other enteropathogens associated with C. parvum was 45·71% for C. perfringens followed by Salmonella spp (40·0%), rotavirus (36·0%), coronavirus (16·0%), E. coli (12·0%) and Eimeria spp (4·0%) The sensitivity for detection of C. parvum by ELISA and mZN staining in comparison to PCR was 97·14% and 72·72%, respectively. An important finding of the study was that C. parvum alone was found in only 10% of the diarrhoeic faecal samples, whereas, majority of the samples (90%) showed mixed infections ranging from a combination of two to five agents. This is the first documentary proof of C. parvum and associated pathogens responsible for severe periurban outbreaks of bovine calf diarrhoea culminating in heavy mortality from Northern India.

Comparative seroprevalence and risk factor analysis of Trypanosoma evansi infection in equines from different agro-climatic zones of Punjab (India).

As parasitaemia is low and fluctuating during the chronic stage of infection, accurate detection of Trypanosoma evansi in blood is difficult. The primary aims of this investigation were to assess for the first time the seroprevalence of T. evansi in all agro-climatic zones of Punjab, by indirect enzyme-linked immunosorbent assay (iELISA) and card agglutination test (CATT/T. evansi), and to evaluate the risk factors associated with latent trypanosomosis. A total of 319 equine serum samples collected from 12 districts of Punjab (India) belonging to different agro-climatic zones revealed 39 (12.23%) and 9 (2.82%) samples to be positive by CATT/T. evansi and iELISA, respectively. The highest prevalence was recorded from the Ludhiana district (42.86% and 7.14% by CATT/T. evansi and iELISA, respectively) in the central plain zone (for which the overall prevalence was 15% and 4.17%, respectively). There was fair agreement between the tests for the detection of T. evansi (kappa = 0.345). Species was the most influential risk factor for infection, with odds ratios (ORs) of 2.81 and 5.63 for donkeys/ mules, in comparison with horses, by CATT/T. evansi and iELISA, respectively. The female equine population (OR = 3.13, 95% confidence interval [CI] = 1.32-7.67 [CATT]) was found to be at a higher risk of seropositivity for T. evansi, particularly on 'unorganised' (inappropriately managed) farms (OR = 3.18, 95% CI = 1.53- 6.65 [CATT]) and among animals used for commercial purposes (OR = 2.51, 95% CI = 1.20-5.21 [CATT]). In conclusion, to declare disease-free status, use of the iELISA followed by retesting of suspect samples by CATT/T. evansi is suggested.

La détection minutieuse de Trypanosoma evansi dans le sang est difficile en raison du nombre faible et fluctuant de parasites pendant la phase chronique de l'infection. L'étude présentée par les auteurs vise, d'une part, à réaliser une première évaluation de la prévalence sérologique de T. evansi dans chacune des zones agro-climatiques du Pendjab en utilisant une épreuve immuno-enzymatique (ELISA) indirecte et le test d'agglutination sur carte pour la trypanosomose (CATT/T. evansi) et, d'autre part, à évaluer les facteurs de risque associés à une présence inapparente de la trypanosomose. Au total, sur les 319 sérums d'équidés prélevés dans 12 districts du Pendjab (Inde) appartenant à des zones agro-climatiques différentes, 39 échantillons (12,23 %) ont donné des résultats positifs avec le CATT/T. evansi et 9 échantillons (2,82 %) ont donné des résultats positifs à l'ELISA indirecte. La prévalence la plus élevée a été enregistrée dans le district de Ludhiana (42,86 % de résultats positifs avec le CATT/T. evansi et 7,14 % de résultats positifs avec l'ELISA indirecte) dans la zone des plaines centrales (où la prévalence globale s'élevait, suivant les méthodes de test, à 15 % et 4,17 %, respectivement). La détection de T. evansi par les deux tests a été concordante (kappa = 0,345). Le facteur de risque ayant le plus d'influence sur la probabilité d'infection était l'espèce, ce risque étant plus élevé chez les ânes et les mulets que chez les chevaux (rapport de cotes [odds ratio, OR] de 2,81 [CATT/T. evansi] et de 5,63 [ELISA indirecte]). Les femelles présentaient également un risque plus élevé de posséder des anticorps anti-T. evansi que les mâles (OR = 3,13 ; intervalle de confiance [IC] à 95 % : 1,32­7,67 [CATT]), en particulier dans les élevages « informels ¼ (sans gestion sanitaire) (OR = 3,18 ; IC à 95 % : 1,53­6,65 [CATT]) ainsi que parmi les animaux utilisés à des fins commerciales (OR = 2,51 ; IC à 95 % : 1,20­5,21 [CATT]). En conclusion, pour la démonstration de l'absence d'anticorps, les auteurs recommandent d'utiliser l'ELISA indirecte puis de soumettre les échantillons douteux à un test de confirmation au moyen du CATT/T. evansi.

La detección precisa de Trypanosoma evansi en la sangre resulta difícil porque en la fase crónica de la infección la parasitemia es baja y fluctuante. Los autores describen una investigación encaminada principalmente a determinar por primera vez la seroprevalencia de T. evansi en todas las zonas agroclimáticas del Punjab por ensayo inmunoenzimático indirecto (ELISAi) y por aglutinación en placa, así como los factores de riesgo asociados a la tripanosomosis latente. De un total de 319 muestras de suero equino procedentes de 12 distritos del Punjab (India) situados en diferentes zonas agroclimáticas, la aglutinación en placa deparó resultado positivo en 39 de ellas (un 12,23%) y el ELISAi en 9 (2,82%). El máximo nivel de prevalencia se registró en el distrito de Ludhiana (42,86% y 7,14% por aglutinación en placa y ELISAi, respectivamente), sito en la zona de la planicie central (que en conjunto deparó una prevalencia del 15% y el 4,17%, respectivamente). Ambas pruebas resultaron bastante coincidentes por lo que respecta a la detección de T. evansi (coeficiente kappa = 0,345). El factor de riesgo más influyente resultó ser la especie: en comparación con los caballos, los asnos o mulas presentaban una razón de probabilidad (RP) de 2,81 y 5,63 para la aglutinación en placa y el ELISAi respectivamente. Se observó que la población de yeguas (RP = 3,13; intervalo de confianza [IC] al 95% = 1,32­7,67 [aglutinación en placa]) presentaba un riesgo más elevado de seropositividad para T. evansi, especialmente en explotaciones «desorganizadas¼ (mal gestionadas) (RP = 3,18; IC 95% = 1,53­6,65 [aglutinación en placa]) y entre los animales utilizados con fines comerciales (RP = 2,51; IC 95% = 1,20­5,21 [aglutinación en placa]). Los autores concluyen proponiendo que, a los efectos de declarar la ausencia de enfermedad, se utilice en primer lugar el ELISAi, seguido de la prueba de aglutinación en placa para las muestras sospechosas.

Seroprevalence of Toxoplasma gondii in South Asian countries.

Toxoplasmosis, a cosmopolitan zoonosis, is caused by an apicomplexan, obligate, intracellular protozoan parasite, Toxoplasma gondii. Nearly all animals, including humans, are at risk owing to its broad geographical distribution. The authors searched published data related to T. gondii in databases, including Google Scholar, PubMed and Science Direct for South Asian countries, and retrieved a total of 113 articles fulfilling the criterion of seroprevalence investigation. Toxoplasma gondii infection in livestock and humans was investigated using various serological tests. In these studies, a total of 14,431 samples from domestic animals and 53,899 samples from humans were screened for anti-T. gondii antibodies in all South Asian countries. Among the animals, cattle (n = 1,981), goats (n = 3,285), buffaloes (n = 1,695), sheep (n = 1,747), cats (n = 1,480), camels (n = 435), elephants (n = 45), pigs (n = 920), dogs (n = 1,604) and poultry (n = 1,206) were tested. This comprehensive review will be useful to biologists, public health workers, physicians and veterinarians and provides a better understanding of the distribution of T. gondii in this region. Furthermore, this knowledge will support efforts to find and apply effective prevention measures to better manage this zoonosis in South Asian countries.

La toxoplasmose est une maladie cosmopolite causée par Toxoplasma gondii, un protozoaire unicellulaire obligatoire appartenant au phylum des Apicomplexa. Du fait de sa distribution géographique, pratiquement toutes les espèces animales sont exposées, ainsi que l'homme. Les auteurs ont fait une recherche dans plusieurs bases de données, dont Google Scholar, PubMed et Science Direct, sur les articles consacrés à T. gondii dans les pays d'Asie du Sud, qui a permis d'extraire un total de 113 articles présentant toutes les caractéristiques d'une enquête sérologique. L'infection par Toxoplasma gondii chez l'homme et chez les animaux d'élevage a fait l'objet de plusieurs enquêtes recourant à divers tests sérologiques. Ces études font état d'un total de 14 431 échantillons prélevés d'animaux domestiques et de 53 899 échantillons prélevés chez l'homme, qui ont été soumis à une épreuve de détection d'anticorps dirigés contre T. gondii dans les pays d'Asie du Sud. Les études ont couvert les espèces suivantes : bovins (n = 1 981), chèvres (n = 3 285), buffles (n = 1 695), moutons (n = 1 747), chats (n = 1 480), chameaux (n = 435), éléphants (n = 45), porcs (n = 920), chiens (n = 1 604) et volailles (n = 1 206). Ce panorama exhaustif sera utile aux biologistes, aux intervenants en santé publique, aux médecins et aux vétérinaires et permettra de mieux appréhender la distribution de T. gondii dans la région. Ces connaissances contribueront à concevoir et à appliquer des mesures de prévention efficaces afin de mieux gérer cette zoonose dans les pays d'Asie du Sud.

La toxoplasmosis es una zoonosis cosmopolita causada por un protozoo, parásito intracelular obligado, del grupo de los apicomplejos: Toxoplasma gondii. Por su amplia distribución geográfica, constituye una amenaza para casi todos los animales, incluido el ser humano. Tras indagar en bases de datos de publicaciones (Google Scholar, PubMed y Science Direct) en busca de información relacionada con la presencia de T. gondii en los países del meridión asiático, los autores encontraron un total de 113 artículos que cumplían el criterio de dar cuenta de investigaciones sobre la seroprevalencia. Para estudiar la infección por Toxoplasma gondii en el ganado y el ser humano se habían empleado diversas pruebas serológicas. En el conjunto de esos estudios, que cubrían todos los países de Asia meridional, se habían analizado un total de 14.431 muestras de animales domésticos y 53.899 muestras humanas para detectar anticuerpos contra T. gondii. Los animales analizados eran: ganado vacuno (n = 1.981), cabras (n = 3.285), búfalos (n = 1.695), ovejas (n = 1.747), gatos (n = 1.480), camellos (n = 435), elefantes (n = 45), cerdos (n = 920), perros (n = 1.604) y aves de corral (n = 1.206). Este repaso general, que resultará útil a biólogos, agentes de salud pública, médicos y veterinarios, permite conocer mejor la distribución de T. gondii en la región, lo que además será de ayuda a la hora de determinar y aplicar medidas eficaces de prevención con objeto de controlar más eficazmente esta zoonosis en los países de Asia meridional.

PCR and ELISA vis-à-vis microscopy for detection of bovine anaplasmosis: a study on associated risk of an upcoming problem in North India.

This investigation demonstrates the status of bovine anaplasmosis caused by A. marginale in bovines from Submountain and Undulating Zone of Punjab. Out of 184 suspected animals, 25 (19.51%), 47 (31.71%), and 78 (68.75%) were positive by microscopy, indirect ELISA, and PCR assay, respectively. The microscopy showed 29% sensitivity and 99% specificity, while ELISA showed 32% sensitivity and 79% specificity in concordance with PCR assay. Five false negative samples by msp1ß PCR were reconfirmed for Anaplasma spp. targeting 16S rRNA gene. The sequence analysis showed the presence for A. marginale specific restriction site, indicating variation in the local strains of the organism resulting in no amplification with msp1ß gene primers. Of 82 samples positive by PCR, 57 were negative by ELISA indicating lower efficacy of ELISA to detect early anaplasmosis. The assessment of risk factor with results of PCR technique indicated that cattle (Odds ratio = 2.884), particularly those of age > 1 years (Odds ratio = 2.204) of district Pathankot (Odds ratio = 3.182) of Submountain Zone (Odds ratio = 2.086), were at high risk of anaplasmosis. All three districts of Submountain Zone are at higher risk indicating the impact of biotic and abiotic factors on the incidence of disease.

Induced neurocysticercosis in rhesus monkeys (Macaca mulatta) produces clinical signs and lesions similar to natural disease in man.

Neurocysticercosis is a serious endemic zoonosis resulting in increased cases of seizure and epilepsy in humans. The genesis of clinical manifestations of the disease through experimental animal models is poorly exploited. The monkeys may prove useful for the purpose due to their behavior and cognitive responses mimicking man. In this study, neurocysticercosis was induced in two rhesus monkeys each with 12,000 and 6,000 eggs, whereas three monkeys were given placebo. The monkeys given higher dose developed hyperexcitability, epileptic seizures, muscular tremors, digital cramps at 10 DPI, and finally paralysis of limbs, followed by death on 67 DPI, whereas the monkeys given lower dose showed delayed and milder clinical signs. On necropsy, all the infected monkeys showed numerous cysticerci in the brain. Histopathologically, heavily infected monkeys revealed liquefactive necrosis and formation of irregular cystic cavities lined by atrophied parenchymal septa with remnants of neuropil of the cerebrum. In contrast, the monkeys infected with lower dose showed formation of typical foreign body granulomas characterized by central liquefaction surrounded by chronic inflammatory response. It was concluded that the inflammatory and immune response exerted by the host against cysticerci, in turn, led to histopathological lesions and the resultant clinical signs thereof.

Development of some larval nematodes in experimental and natural animal hosts: an insight into development of pathological lesions vis-a-vis host-parasite interactions.

Infective third-stage larvae of three spiruroid nematodes, Ascarops strongylina and Physocephalus sexalatus of pigs and Spirocerca lupi of dogs, were recovered from 14 species of coprophagous beetles belonging to 4 different genera. These larvae were fed to rabbits and/or guinea pigs to study their development in these experimental hosts. Larvae of A. strongylina reached the adult stage in all rabbits and one guinea pig. The adult worms recovered in these hosts were 40% and 4%, respectively, and became diminutive in comparison to their natural hosts. The larvae of P. sexalatus became reencysted in the gastric wall of rabbits inducing marked pathological changes. The infective larvae of S. lupi became reencapsulated in the stomach wall of the rabbit and also showed development in the aortic wall. Adults of Toxocara canis of dog, collected from 5 different regions of the Indian subcontinent, varied significantly in size. The mouse passage of infective larvae of one of these types led to the recovery of the adults from the experimental dogs that were smaller in size and caused severe pathology in natural experimental hosts. Developmental effects shown in experimental hosts and host specificity are of value in understanding the evolution of nematode parasitism.

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.

Comparative evaluation of various diagnostic techniques for detection of Cryptosporidium infection from the faecal samples of diarrhoeic bovine calves.

Background: Cryptosporidium, an opportunistic, zoonotic, apicomplexan parasite, is one of the most common causes of diarrhea in neonatal bovine calves around the globe. Bovine calves act as a major source of infection by excreting huge numbers of highly resistant oocysts in faeces, which can survive for a long time in extreme environmental conditions. As low as ten oocysts can cause disease and mortality, leading to the requirement of an early and accurate diagnosis for proper and favorable prognosis, management, and control. Aims: The current study was conducted with the objective to evaluate various diagnostic techniques (acid fast staining, negative staining, fluorescent, ELISA, PCR, nested PCR, and qPCR) for the detection of Cryptosporidium in the faecal samples of diarrheic bovine calves. Methods: Two hundred diarrheic faecal samples from bovine calves were collected and subjected to these techniques for Cryptosporidium diagnosis. Results of these were evaluated for diagnostic comparison. Results: Out of 200 faecal samples evaluated, 24% (48/200) were detected positive for Cryptosporidium using a combination of two techniques as gold standard criteria. Cohen's kappa value indicated moderate to almost perfect agreement (0.616 to 0.986) among all the techniques used in the present study. Leishman staining showed the lowest sensitivity (54.17%), while nested PCR and qPCR showed the highest sensitivity (97.92%). Diagnostic specificity of all these tests ranged from 98.68 to 100%. Conclusion: Auramine stain was used for the first time in the bovine calves in India for the detection and diagnostic comparison of Cryptosporidium. It showed strong agreement with the molecular as well as classical diagnostic techniques, and can be used for primary screening for better diagnosis.

Molecular characterization and histo-physiological alterations induced by concurrent helminthosis in the liver of urban commensal rodents in Punjab, India.

BACKGROUND: Rodents harbour a number of parasites of public health importance, thus, they threaten human health and livestock. AIMS: The present study aimed to characterize two helminthic species found in commensal rodents and record histo-physiological alterations induced by them. METHODS: A total of 300 synanthropic rodents of three species: Rattus rattus (n=201), Bandicota bengalensis (n=90), and Mus musculus ( n=09 ) were live trapped and necropsied in different seasons during November 2017 to October 2019 at Ludhiana, Punjab, India. RESULTS: Liver of two species B. bengalensis (72.22%) and R. rattus (65.67%) were found infected with two helminthic parasites Taenia taeniaeformis, and Calodium hepaticum. These endoparasites were present either alone (4.33-6.33%) or as mixed infection (65.55%). The level of total proteins and liver marker enzymes including aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were found significantly higher in the liver of rodent species infected with single and mixed infection compared to those with no infection. In histopathological assay, granulomatous liver lesions and necrosis of hepatocytes were seen which were associated with eggs and adults of C. hepaticum and inflammatory reaction in hepatic parenchyma adjoining to cysts of T. taeniaeformis. Based upon scanning electron microscopy (SEM) identification and molecular characterization using mitochondrial cytochrome oxidase I (COI) region, the metacestodes in whitish cysts were confirmed to be of T. taeniaeformis for the first time in Punjab, India. CONCLUSION: The study highlights an alarmingly high infection of rodents with zoonotic parasites and suggests immediate pest (rodent) control to check the dissemination of zoonotic diseases by helminth species under study.

Spatial seroepidemiology, risk assessment and haemato-biochemical implications of bovine trypanosomiasis in low lying areas of Punjab, India.

A cross-sectional study was carried out on 594 bovines (341 buffalo adults, 31 buffalo calves, 163 cattle adults, and 59 cattle calves) to assess the exposure of native bovine population to T. evansi elicited trypanosomiasis in the low-lying areas of Punjab (India). We ruled out the endemicity of the disease with 10.77% (95%CI = 8.53-13.52) sero-positive and 23.56% (95%CI = 20.33-27.15) suspected cases by card agglutination assay. We have presented the spatial distribution of these cases as a guideline to local veterinary practitioners and policy-makers. The categorical assessment of risk factors revealed buffalo adults are the most susceptible group in the state despite insignificant differences in farm management practices. A significant increase in the WBC, platelet, AST and serum iron, and decrease in haemoglobin, haematocrit volume, and serum glucose was recorded in both T. evansi positive and suspected animals.

Sero-prevalence and risk factor analysis of Theileria equi infection in equids from different agro-climatic zones of Punjab (India) by Indirect Immunofluorescence Antibody test.

In the Indian sub-continent, equine piroplasmosis (EP) is an imperative tick-borne disease of equids predominately caused by obligatory intra-erythrocytic protozoa Theileria equi. Present exploration is the first epidemiological survey report based on Indirect Immunofluorescence Antibody test (IFAT). A total of 120 equines (98 horses and 22 donkeys/mules) from five districts of two main agro-climatic zones of Punjab were screened. The overall prevalence was 58.33%, with western plane zone at higher infection risk for T. equi (74.50%, OR = 7.45, 95% CI = 62.24-85.76); followed by western zone (46.38%, OR = 4.63, 95% CI = 34-57.7). District wise highest and lowest prevalence was recorded in district Fazilka (80.76%) and Bathinda (35.29%) respectively. IFAT revealed elevated risk of infection of T. equi in the farms with inappropriate management structure. The risk factor connected with the type of host species had an odds ratio of 1.67 for donkeys/mules versus horses for T. equi infection. Followed by presence/absence of tick on animal. This study reveals the endemicity of EP in Punjab with prominence of various odds of infection, associated with latent infection of T. equi detectable by serological techniques as it was noticed that western plane zone and western zone are more prone to EP infection.

Unraveling cryptic epizootiology of equid trypanosomosis in Punjab state of India by parasitological and sero-molecular techniques.

To unravel equid trypanosomosis caused by Trypanosoma evansi in Punjab state of India, a cross sectional study was designed by utilizing parasitological and sero-molecular tools with objective to assess the prevalence of T. evansi in association with various risk factors in all agroclimatic zones of Punjab state of India. Parasitological Romanowksy stained thin blood smears (RSTBS) to detect patent infection, molecular techniques polymerase chain reaction I (PCR I; TBR 1/2 primers; targeting minichromosomal satellite DNA of T. evansi), polymerase chain reaction II (PCR II; TR 3/4 primers; targeting variable surface glycoprotein region DNA of T. evansi) & LAMP (Loop mediated isothermal amplification) assay to detect latent infection and serological assays card agglutination test (CATT/T. evansi) & ELISA (Enzyme linked immunosorbent assay) to detect exposure status of trypanosomosis were utilized in the present study. A total 429 equid blood and serum samples from all the five agroclimatic zones of Punjab state tested by these techniques showed a prevalence of 1.39% (CL: 0-15.28) by RSTBS, 6.52% (10.94-45.09) by both TBR 1/2 PCR and LAMP assay, 5.82% (11.57-38.42) by TR 3/4 PCR, 15.15% (36.57-135.42) with CATT/T. evansi and 22.84% (17.77-840.22) with ELISA. Interpretation of various risk factors revealed that the donkey/mules population (RR = 5.46, 95% [CI] = 0.15-15.56) was found to be at higher risk of T. evansi infection predominantly at 'unorganized' farms (RR = 4.06, 95% [CI] = 0.12-4.51). Animal used for commercial purposes (RR = 3.25, 95% [CI] = 0.06-7.42), rearing of equids with other domestic animals (RR = 2.36, 95% [CI] = 0.10-17.11) and farms without application of fly repellant/insecticides/net (RR = 3.68, 95% [CI] = 0.08-5.94) made them more prone to the disease. This comprehensive report utilizing the classical, serological and molecular diagnostic tools for epidemiology of T. evansi establishes the endemic stability of this infection in all agro climatic zones of Punjab with LAMP assay to be a promisingly sensitive and specific technique for the diagnosis of T. evansi under isothermal conditions in field situations.

Detection of anthelmintic resistance in sheep and goat against fenbendazole by faecal egg count reduction test.

Anthelmintic resistance against commonly used anthelmintic fenbendazole was evaluated by employing faecal egg count reduction test (FECRT) in naturally occurring gastrointestinal (GI) nematodes in the semi organized sheep and goat farms of Ludhiana and Amritsar districts. A total of 80 animals (20 each for sheep and goat in both districts) were randomly selected and their faecal samples were examined by qualitative and quantitative parasitological techniques. Results indicate presence of high level of resistance against fenbendazole in both sheep and goat population of Ludhiana and Amritsar districts. More resistance was observed in the GI nematodes from animals reared in Amritsar district as compared to Ludhiana district. The level of anthelmintic resistance observed was apparently more in sheep than goats.

Prevalence of gastrointestinal parasitism in small ruminants in western zone of Punjab, India.

AIM: The aim of this study was to explore the prevalence of gastrointestinal parasitism in small ruminants in relation to various risk factors in the western zone of Punjab. MATERIALS AND METHODS: During the study, 603 fecal samples (391 of sheep and 212 of goats) were examined qualitatively by floatation and sedimentation techniques, and quantitatively by McMaster technique. RESULTS: Out of the 603 fecal (391 sheep and 212 goats) samples examined, 501 were found positive for endoparasitic infection with an overall prevalence of 83.08%, consisting of 85.16% and 79.24% in sheep and goats, respectively. Egg per gram in sheep was apparently more 1441.88±77.72 than goats 1168.57±78.31. The associated risk factors with the prevalence of gastrointestinal tract (GIT) parasites showed that females (85.97%) were significantly more susceptible than males (69.23%). Age wise the adults (>6 months) were significantly more prone to parasitic infection as compared to young ones (<6 months). Seasonal variation was recorded throughout the year and was significantly highest during monsoon (90.10%), followed by winter (83.84%) and summer (78.35%). CONCLUSION: The study revealed an overall prevalence of 83.08% of GIT parasitic infections in small ruminants constituting 85.16% in sheep and 79.24% in goats in the western zone of Punjab. The most relevant risk factors for the prevalence of gastrointestinal parasitism in ruminants were sex, age, and season.

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 survey in relation to risk factors and haemato-biochemical alteration in Theileria equi infection of equines in Punjab Province, India.

Equine piroplasmosis caused by Theileria equi, an apicomplexan intracellular haemoprotozoan parasite effects equids throughout the world. Current investigation is the first detailed epidemiological survey report based on conventional (blood smear) and primary polymerase chain reaction (PCR) for the detection of T. equi on 464 equids (426 horses and 38 donkeys/mules) in Punjab province of India. PCR targeting 18S rRNA gene of T. equi produced high fidelity 709bp amplification products with 100% concordance with blood films. The prevalence of infection was proportional (P≤0.01) to temperature and aridness of the regions, which turned out to be the most important physical factor pertaining to T. equi infection. Spatial distribution analysis revealed an increasing trend of disease prevalence from north-eastern to south-western region of Punjab by both the techniques. Haemato-biochemical analysis revealed significant decrease in total erythrocyte count and haemoglobin; and increase in mean corpuscular haemoglobin, serum globulin, glucose, creatinine, aspartate aminotransferase and bilirubin levels (P≤0.05). This study divulges the endemicity of equine theileriosis in Punjab with the prominence of various odds of infection, emphasising the pathophysiological manifestation associated with latent infection of T. equi detectable by PCR.

Validation of Romanowsky staining as a novel screening test for the detection of faecal cryptosporidial oocysts.

Cryptosporidiosis is an emerging waterborne protozoan disease and one of the major causes of neonatal diarrhea in humans and animals. But the disease remains under diagnosed due to lack of availability of special stains in majority of laboratories at primary health centers. Therefore, it requires a rapid screening test for routine diagnosis in conventional laboratory set up. In this pursuit, the present study was planned. During this study, fecal samples from 100 representative animals randomly selected from 17 out breaks of bovine calf diarrhea, were stained with modified Ziehl Neelsen staining (mZN) and Leishman's stain to demonstrate cryptosporidial oocysts and for routine fecal examination, respectively. By mZN staining, 25 cases confirmed the presence of cryptosporidial oocysts. However, examination of Leishman's stained fecal smears revealed round hollow unstained bodies resembling cryptosporidia in 20 cases. Therefore, a comparative morphometric analysis was made between the two techniques to determine their relative efficacy in demonstrating cryptosporidia in the feces of affected animals. The analyses showed that the Leishman's stain can be effective in making a presumptive diagnosis of cryptosporidiosis with a little experience. Confirmation of cryptosporidiosis was done by histopathological examination of intestinal sections of calves died during these out breaks. The findings appear to have great clinical value for routine laboratory screening of fecal samples for cryptosporidiosis as conventional Romanowsky stains are readily available and used for multipurpose examination in most of the laboratories at grass root level. Perusal of literature proved this to be the first attempt at easy diagnostics for cryptosporidiosis.

Patho-physiological observations in natural concurrent infections of helminth parasites of zoonotic importance in the wild rodents, Bandicota bengalensis.

Screening of 83 mature wild rodents of Bandicota bengalensis (52 male and 31 females), live trapped from premises near railway station, fish market and agricultural fields in Ludhiana district of Punjab province, India was carried out to assess the natural occurrence of helminth parasites and their potential as reservoirs of zoonoses. On necropsy, intestine of rats was found infected with adult cestode parasites of Hymenolepis diminuta and H. nana. Liver was found infected with Capillaria hepatica and Cysticercus fasciolaris. These parasites were present either alone or as concurrent infection. In overall, 68.67 % rats were found infected of which 59.65 % were infected with nematode parasite, C. hepatica and 75.44 % with cestode parasites. Grossly, pathognomonic lesions of C. hepatica and C. fasciolaris were seen clearly and microscopic changes were observed only in severely infected cases. Overall investigation on the effect of these parasitic infections on pathophysiology of the host revealed that rodents serve as reservoirs of these parasitic infections without having much pathogenic effect on their vital organs, fecundity and enzyme activities. Because of its close association with human habitations, B. bengalensis may act as an important source of zoonotic infections and thus direct or indirect contact with excrements and carcasses of this rodent species, should be avoided.