Duplex real-time PCR methods for molecular detection and characterization of canine tick-borne haemoparasites from Punjab state, India.

BACKGROUND: Microscopy is a routinely used technique for the diagnosis of canine tick-borne haemoparasitic diseases in various clinical laboratories worldwide. In an attempt to provide better diagnostic assay to the clients for effective management of these diseases duplex real-time PCR assays were applied. METHODS AND RESULTS: Blood samples (n = 338) aseptically collected from suspected dogs of Central Plain Zone of Punjab state, India were subjected to SYBR Green based real-time duplex PCR assays for simultaneous detection of B. vogeli & E. canis and B. gibsoni & H. canis. Results revealed an overall prevalence rate of canine tick-borne haemoparasites as 54.1%, amongst which H. canis was the predominant (25.4%), followed by B. gibsoni (16.3%), E. canis (10.7%) and B. vogeli (1.8%). Sensitivity and specificity of the duplex assays ranged from 59.04 to 100.0% and 58.12 to 92.52%, respectively and their strength of agreement was ″fair″ with kappa value statistics. A significant (p < 0.05) association between prevalence of B. gibsoni, H. canis and E. canis infection with risk factors like sex, breed, season and location was recorded. The ancestral background of the field isolates of haemoparasites was also studied by phylogenetic analysis of their nucleotide sequences. CONCLUSIONS: SYBR Green dye based duplex real-time PCR assays proved to be highly sensitive, specific, rapid and affordable diagnostic tests for use by clinicians to save the life of pets.

In vitro acaricidal activity of Piper longum L. against amitraz resistant Rhipicephalus microplus (Acari: Ixodidae).

The cattle tick, Rhipicephalus microplus Canestrini (Acari: Ixodidae) is one of the most important tick species severely affecting health and causes huge losses to dairy industry. Chemical acaricides are mainly applied for tick control but development of resistance, environmental pollution and contamination of milk and meat products with residues has led to exploration alternative eco-friendly tick control strategies. The dried fruits of Piper longum L. (Indian long pepper, Thippali or Pippali) generally used as flavoring agent have also been shown to have insecticidal property. Different concentrations (0.625%-10%) of alcoholic and aqueous extracts of Piper longum L. were prepared and evaluated for acaricidal activity against amitraz resistant R. microplus adult and larval stages. Against larval stages a dose-dependent mortality response was recorded for both extracts and higher acaricidal property was exhibited by the alcoholic extract with LC50 and LC95 (95% CL) values of 0.488% (0.48-0.49) and 1.39% (1.35-1.44), respectively. Similarly, against adult engorged females, ethanolic extract showed higher acaricidal property with LC50 and LC95 (95% CL) values of 4.67% (4.61-4.74) and 12.38% (12.05-12.73), respectively. Significant (p < 0.05) reduction was recorded in reproductive index of ticks treated and but no effect on hatchability of eggs was recorded in treated groups. The present study establishes acaricidal activity of P. longum fruit extracts against both larval and adult stages of amitraz resistant population of cattle tick.

In vitro assessment of synergistic combinations of essential oils against Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

Rhipicephalus (Boophilus) microplus (Acari: Ixodidae), is one of the most important ectoparasite of cattle, responsible for causing severe economic losses in the tropical and subtropical regions of the world. The development of resistance to most of the commonly used chemical acaricides has stimulated the search for new herbal products as an eco-friendly tick control alternative. The aim of the present study was to evaluate the in vitro acaricidal activity of essential oils (EOs) of cloves (Syzygium aromaticum), bark of cinnamon (Cinnamomum zeylanicum) and leaves of lemon grass (Cymbopogon citratus) against unfed larvae of R. (B.)microplus by larval packet test. Also, various combinations were prepared by mixing of equal concentrations of any two and all three EOs and used to study the effects of interactions of mixtures against larvae of R. (B.) microplus. The lethal concentrations at 50% (LC50), 90% (LC90) and 99% (LC99) with 95% confidence limits (CL) were estimated by Probit analysis using PoloPlus. The computer software, CompuSyn, was used for determining the effects of interactions (synergistic, additive or antagonistic) of EO mixtures by calculation of Combination index (CI) and Dose-reduction index (DRI). Among the individual and mixture of two EOs, cinnamon EO and cinnamon EO + lemon grass EO combination showed highest acaricidal activity against R. (B.) microplus larvae. Further, the combination of cinnamon EO + lemon grass EO showed high synergism with CI value of 0.381, followed by cinnamon EO + clove EO showing moderate synergism whereas, clove EO + lemon grass EO showed only additive effect, with favorable dose reduction for each constituent drug in all three combinations. The combination of all three EOs (1:1:1) showed high synergism (CI value of 0.376) and favorable dose reduction (DRI index of 8.19, 25.64 and 4.64 for clove, cinnamon and lemon grass EOs, respectively) against R. (B.) microplus larvae.

In vitro assessment of the acaricidal activity of Piper longum, Piper nigrum, and Zingiber officinale extracts against Hyalomma anatolicum ticks.

Ticks and tick-borne diseases are a major constraint for the sustainable cattle industry in the tropical and subtropical regions including the Indian subcontinent. The development of resistance to most of the commonly used acaricides leads to an attempt to screen plant extracts and their combinations for their possible acaricidal activity to develop an eco-friendly tick control alternative. An alcoholic and various aqueous extracts of Piper longum, Piper nigrum and Zingiber officinale and their combinations were evaluated for acaricidal activity against the three-host ixodid tick, Hyalomma anatolicum by larval immersion test using 14-21 days old unfed larvae. The efficacy was assessed by measuring larval mortality (%) and the lethal concentrations for 50% (LC50) and 95% (LC95) with their 95% confidence limits (CL) values were estimated by applying regression equation analysis to the probit transformed data of mortality. A concentration-dependent mortality response was recorded in all extracts prepared from seeds of P. longum and P. nigrum and their combinations. The highest acaricidal property was exhibited by the alcoholic extract of P. longum seeds with the minimum LC50 and LC95 (95% CL) values of 0.071% (0.07-0.072) and 0.135% (0.13-0.14), respectively, followed by alcoholic combinations. Interestingly, no acaricidal activity was recorded in extracts prepared from the rhizome of Z. officinale. The results indicated that the ethanolic extracts of P. longum and P. nigrum and their combinations can be used effectively for tick control in an integrated format.

In vitro acaricidal activity of Atropa belladonna and its components, scopolamine and atropine, against Rhipicephalus (Boophilus) microplus.

In vitro efficacy of methanolic extract of Atropa belladonna and its components scopolamine and atropine was assessed against Rhipicephalus (Boophilus) microplus. Five concentrations of the extract (1.25%, 2.5%, 5%, 10%, and 20%) were used whereas scopolamine and atropine were each tested at 0.1%. In adult immersion test, the extract was lethal to ticks at 20% concentration. The LC50 and LC95 values were determined as 6.875% and 17.306%, respectively. The extract caused a significant reduction (P < 0.05) in egg mass production at 10% concentration. In larval packet test, the extract was lethal to larvae in the concentrations of 10% and 20% after 24 h, with LC50 and LC95 values of 1.321% and 4.935%, respectively. Scopolamine and atropine showed 93.3% and 60.0% mortality of adult ticks, respectively, but they caused complete (100%) blocking of hatching as well as 100% larval mortality. Scopolamine and atropine were observed to be more potent than the crude extract at an equivalent concentration in both the bioassays.

Molecular Survey of Tick-Borne Haemoparasites of Dogs by Multiplex Polymerase Chain Reaction from Punjab, India.

PURPOSE: Tick-transmitted parasites as Babesia gibsoni, Babesia vogeli, Ehrlichia canis, and Hepatozoon canis are major health concern for dogs. Owing to prevalence and infection severity, there is need of sensitive, specific, and affordable test for their simultaneous detection. METHODS: Prevalence of B. gibsoni, B. vogeli, E. canis, and H. canis infections was assessed on 719 blood samples by microscopy and multiplex PCR assay targeting 18S rRNA (B. gibsoni & H. canis), ITS1 & 5.8S rRNA (B. vogeli) and VirB9 gene (E. canis). An internal control (canine-actin) was also included to increase the accuracy of assay and effect of associated risk factors with disease prevalence was also studied. RESULTS: Microscopic prevalence of B. gibsoni, B. vogeli, E. canis and H. canis was 5.0%, 0.1%, 1.4% and 1.0%, respectively, whereas with multiplex PCR assay, the corresponding values were 8.9%, 1.1%, 2.6% and 5.1% besides concurrent infections of B. gibsoni & H. canis (0.4%), B. gibsoni & E. canis (0.4%), E. canis & H. canis (0.3%) and B. gibsoni & B. vogeli (0.1%). Analytical sensitivity of developed assay was 0.1pg (B. gibsoni & H. canis), 0.01pg (B. vogeli), and 1.0pg (E. canis). A ″fair″ (B. vogeli & H. canis) to ″substantial″ (B. gibsoni & E. canis) agreement between two tests was observed with data as statistically significant. Breed, sex and location were significantly associated with B. gibsoni infection. CONCLUSION: The developed multiplex PCR assay offers a potential solution to detect these pathogens simultaneously, aiding in timely diagnosis and effective disease management in suspected dogs.

Discrimination, molecular characterisation and phylogenetic comparison of porcine Eimeria spp. in India.

Infections with Eimeria spp. are common in pigs worldwide, occasionally affecting animals clinically after weaning or during the fattening period when diarrhoea and weight loss can be observed upon infection with the more pathogenic species. Molecular characterization of pathogens is valuable to accurately delimit species and development novel diagnostics, although sequences which define Eimeria species that infect pigs are scarce. Only three of the eight common species are currently represented in GenBank. In this study we describe the occurrence of Eimeria species in pigs sampled in Punjab, India; going on to use the samples to generate new species-specific 18S rDNA sequences for all of the previously uncharacterised species. Using these data we report the first phylogenetic analyses to include the eight Eimeria species that commonly infect the domestic pig (Sus scrofa domesticus). Consideration of phylogenetic trees produced using Maximum Likelihood, Neighbour Joining, Maximum Parsimony and Unweighted Pair Group Method with Arithmetic Mean methods indicate that the 18S rDNA sequences present lower levels of genetic diversity than Eimeria which infect avian species and are insufficient to infer stable phylogenies.

In-vitro efficacy of a botanical acaricide and its active ingredients against larvae of susceptible and acaricide-resistant strains of Rhipicephalus (Boophilus) microplus Canestrini (Acari: Ixodidae).

Ticks and tick-borne diseases are a major constraint for a sustainable cattle industry in the tropical and subtropical regions of the world. The development of resistance to most of the commonly used acaricides has led to an assessment of herbal products for acaricidal activity as an eco-friendly tick control alternative. A botanical product, Essentria® IC-3 insect concentrate containing rosemary oil (10%), geraniol (5%) and peppermint oil (2%), acts on target pests by blocking octopamine. Essentria® IC-3 and its active components were evaluated for larvicidal activity against several susceptible and acaricide-resistant strains of the cattle fever tick, Rhipicephalus (Boophilus) microplus Canestrini by Larval Packet test using 14-21 d old unfed larvae. The efficacy was assessed by measuring percent larval mortality and estimating lethal concentrations at 50% (LC50) and 95% (LC95) with 95% confidence limits (CL) using probit analysis. The LC50 and LC95 (95% CL) values for Essentria® IC-3 against the susceptible strain were estimated as 0.647% (0.59-0.69) and 1.033% (0.94-1.19), respectively, whereas, LC50 and LC95 values for other strains were variable, ranging from 0.597-1.674% and 0.927-2.236%, respectively. Among the various active ingredients, the larvicidal property of Essentria® IC-3 seem to be attributable mainly to geraniol and the LC50 and LC95 (95% CL) values for geraniol against the susceptible Deutch strain were estimated as 0.656% (0.61-0.69) and 1.114% (1.03-1.25), respectively. The comparison of LC50 and LC95 values of acaricide-resistant strains showed susceptibility comparable to Deutch against geraniol except for the Las Palmas strain. We report a low level of resistance in some of the acaricide-resistant strains against the herbal acaricide in the cattle tick for the first time, possibly due to cross-resistance to chemical acaricides.

Comparison of natural and artificial odor lures for nilgai (Boselaphus tragocamelus) and white-tailed deer (Odocoileus virginianus) in South Texas: Developing treatment for cattle fever tick eradication.

Cattle fever ticks (CFT), vectors of bovine babesiosis and anaplasmosis, were eradicated from the United States by 1943, but are frequently reintroduced from neighboring border states of Mexico via stray cattle and wildlife hosts including white-tailed deer (Odocoileus virginianus) (WTD) and nilgai antelope (Boselaphus tragocamelus). Nilgai antelope are exotic bovids from India that are hosts of CFT, have large home ranges as compared to WTD, thus have the potential to spread CFT through the landscape. Currently, there are no methods to control CFT on nilgai. Odor lures were evaluated to determine if nilgai could be attracted to a central point for development of control methods. Four treatments, nilgai offal a natural odor lure was used as the positive control; and compared to three artificial odors; screw worm lure, volatile fatty acids, citronella oil. Studies were conducted on a free-ranging population of nilgai at the East Foundation's Santa Rosa Ranch (Kenedy Co., near Riviera, Texas, USA). Game cameras were used to document visitation to the lures. In the ten randomly placed transects, 110 nilgai and 104 WTD were photographed. Offal had significantly more visits by nilgai (71% of total visits) than screwworm (15%), VFA (11%), and citronella (4%). For WTD, there was no significant difference in visitation at the lure treatments.

Movement patterns of nilgai antelope in South Texas: Implications for cattle fever tick management.

Wildlife, both native and introduced, can harbor and spread diseases of importance to the livestock industry. Describing movement patterns of such wildlife is essential to formulate effective disease management strategies. Nilgai antelope (Boselaphus tragocamelus) are a free-ranging, introduced ungulate in southern Texas known to carry cattle fever ticks (CFT, Rhipicephalus (Boophilus) microplus, R. (B.) annulatus). CFT are the vector for the etiological agent of bovine babesiosis, a lethal disease causing high mortality in susceptible Bos taurus populations and severely affecting the beef cattle industry. Efforts to eradicate CFT from the United States have been successful. However, a permanent quarantine area is maintained between Texas and Mexico to check its entry from infested areas of neighboring Mexico states on wildlife and stray cattle. In recent years, there has been an increase in CFT infestations outside of the permanent quarantine area in Texas. Nilgai are of interest in understanding how CFT may be spread through the landscape. Thirty nilgai of both sexes were captured and fitted with satellite radio collars in South Texas to gain information about movement patterns, response to disturbances, and movement barriers. Median annual home range sizes were highly variable in males (4665ha, range=571-20,809) and females (1606ha, range=848-29,909). Female movement patterns appeared to be seasonal with peaks during June-August; these peaks appeared to be a function of break-ups in female social groups rather than environmental conditions. Nilgai, which reportedly are sensitive to disturbance, were more likely to relocate into new areas immediately after being captured versus four other types of helicopter activities. Nilgai did not cross 1.25m high cattle fences parallel to paved highways but did cross other fence types. Results indicate that females have a higher chance of spreading CFT through the landscape than males, but spread of CFT may be mitigated via maintenance of cattle fences running parallel with paved highways. Our results highlight the importance of documenting species-specific behavior in wildlife-livestock interfaces that can be used to develop effective disease management strategies in the United States and worldwide.

A comparative study on cypermethrin resistance in Rhipicephalus (Boophilus) microplus and Hyalomma anatolicum from Punjab (India).

A study to evaluate cypermethrin resistance in Rhipicephalus (Boophilus) microplus and Hyalomma anatolicum collected from Muktsar and Mansa districts of Punjab state, India, was conducted by using adult immersion test (AIT). The regression graphs of probit mortality of ticks plotted against log values of concentrations of cypermethrin was utilized for the determination of slope of mortality, lethal concentration for 50% (LC50), and the resistance factor (RF). On the basis of the data generated on variables (mortality, egg mass weight, reproductive index, and percentage inhibition of oviposition), the resistance levels were categorized. Resistance to cypermethrin was categorized as level II and I in R. (B.) microplus collected from Muktsar and Mansa districts, respectively, whereas, H. anatolicum from both locations showed a susceptible status. The RF values of Muktsar and Mansa field samples of engorged R. (B.) microplus (5.48 and 2.18, respectively) were much higher as those of engorged H. anatolicum (1.12 and 0.82, respectively) indicating a lower level and slower rate of development of cypermethrin resistance in multi-host ticks. The data generated in the current study might be of immense help in formulating suitable control measures against ticks and tick-borne diseases of animals.

Molecular detection of Anaplasma marginale infection in carrier cattle.

Anaplasma marginale infections are responsible for widespread morbidity and mortality particularly in crossbred and exotic breeds of cattle in the tropics and subtropics. In the present study, a semi-nested PCR assay was utilized for the detection of A. marginale infection in carrier cattle from different areas of Punjab state, India. An amplicon of 458 bp specific for msp5 of A. marginale was detected in 45.2% of blood samples when subjected to primary PCR assay against the routine blood smear examination, which revealed inclusion bodies in only 12.5% of samples. Semi-nested PCR employing product of samples negative by primary PCR produced the amplicons of desired size (345 bp) in 51% (29/57) of samples indicating that nested PCR, when coupled with primary PCR, resulted in increased sensitivity in detection of A. marginale infection in carrier cattle from 45.2 to 73.1%. These results suggest higher threshold detection limits of the nested PCR over the conventional technique used for diagnosis of anaplasmosis which is ideally suited for diagnosis of carrier cases which act as nidus for the spread of the infection to the susceptible stock in endemic areas.