High-throughput sequencing of Fasciola spp. shows co-infection and intermediate forms in Balochistan,but only Fasciola gigantica in the Punjab province of Pakistan

Fasciola gigantica and Fasciola hepatica are digenetic trematodes causing fasciolosis in ruminants. The host and geographical distribution of both Fasciola species are influenced by environmental and climatic conditions favouring survival and development of free-living stages and intermediate hosts, and livestock management practices. The aim of the present study was to describe the host distribution of the two Fasciola species in buffalo, cattle, goats, and sheep in the Balochistan and Punjab provinces of Pakistan. 359 flukes were collected from a total of 32 livers from the four livestock species. Deep amplicon sequencing of the internal transcribed spacer region 2 of ribosomal DNA (rDNA ITS-2) and mitochondrial nicotinamide adenine dinucleotide dehydrogenase 1 (mtDNA ND-1) loci confirmed co-infection of F. hepatica and F. gigantica in Balochistan and single species F. gigantica infection in Punjab. In Balochistan, co-infections and hybrids of both Fasciola species were identified in cattle, with more F. hepatica detected than F. gigantica. However, F. hepatica was the only species identified in goats, and F. gigantica was the only species identified in buffalo. In Punjab, all flukes were confirmed as F. gigantica in each of the four livestock species. Overall, the results indicate differences in the host and geographical distribution of F. gigantica and F. hepatica, and provide useful knowledge for the development of control strategies for livestock and humans.     

West Nile virus outbreak in horses,southern France, 2000: results of a serosurvey

During late summer and autumn 2000, a West Nile fever outbreak in southern France resulted in 76 equine clinical cases; 21 horses died. We report the results of a large serosurvey of all equines within a 10-km radius of laboratory-confirmed cases. Blood samples were obtained from 5,107 equines, distributed in groups of 1 to 91 animals. West Nile virus immunoglobulin (Ig) G antibodies were found in 8.5% of animals (n=432). Forty-two percent of the IgG-positive animals were also IgM positive. Horses living in small groups were more affected than those in large groups. The results suggest that West Nile virus is not endemic in the affected area, the Camargue; rather, sporadic outbreaks are separated by long silent periods.     

West Nile virus surveillance, Guadeloupe, 2003-2004

We conducted extensive surveillance for West Nile virus infection in equines and chickens in Guadeloupe in 2003-2004. We showed a high seroprevalence in equines in 2003 related to biome, followed by a major decrease in virus circulation in 2004. No human or equine cases were reported during the study.     

Prolonged incubation period of imported P. vivax malaria in London

Between January 1976 and July 1979, 453 cases of malaria were seen at Hillingdon Hospital. The majority of cases were Plasmodium vivax infections in Asians from the Punjab in Northern India–either new immigrants or United Kingdom resident Asians returning from holidays. Twenty-four cases were contracted in Africa or the Middle East. Figures are presented showing a considerable increase in cases during the period of study. In P. vivax infections the time interval between arrival and development of the acute illness was significantly greater for those subjects entering the United Kingdom in autumn or winter.     

Comparative digestion of timothy (Phleum pratense) fibre by ruminants, equines and rabbits

1. The abilities of cattle, sheep, goats, equines and rabbits to digest mature timothy (Phleum pratense) hay were compared. Apparent digestibilities were partitioned into true digestibility, metabolic faecal output (MFO) and fibre digestibility. The aid of the study was to determine the relative effects of fermentation site (among groups) and of body-weight (within groups) on the efficiency of digestion. 2. The ruminants were superior to equines, which were in turn superior to rabbits, in digesting fibre-components of the hay. A large individual variation in digestibility was noted only for the equines. Increasing body-weight was associated with higher digestibility in ruminants, but no such trends were seen in the non-ruminants. 3. The MFO expressed as a proportion of dry matter intake gave similar values for all groups (0.085-0.118). As a proportion of available microbial substrate originating from the feed, the values were found to be 0.167 for the ruminant, 0.425 for the equines and 2.13 for the rabbits. The value for the rabbits shows that their lower tract microflora must obtain energy from non-fibre components of the feed. No appreciable digestion of the generated microbes by the host was suggested by the values obtained for the equines.     

Female-Headed Households Associated with Lower Childhood Stunting Across Culturally Diverse Regions of Pakistan: Results from a Cross-Sectional Household Survey

Objectives Early childhood stunting adversely influences long-term cognitive and health outcomes. There is limited evidence on whether female empowerment within households could reduce its prevalence. We investigated this relationship in Punjab, Pakistan, which has high stunting rates and a sizeable proportion of female-headed households, and whether this relationship differed across three provincial regions with diverse cultural attitudes towards the role of women in society. Methods Using cross-sectional data from the 2011 Multiple Indicator Cluster Survey, we identified 13,412 children aged 1–4 from 8985 two-parent households in three culturally distinct regions in Punjab, Pakistan. Logistic regression models assessed whether the likelihood of stunting was associated with female-headed households, a proxy for female empowerment, and whether this relationship differed by region. Regressions controlled for child- and household-level covariates. Results Children had 26% lower odds of stunting among female-headed households (OR 0.74, CI 0.60, 0.90). The interaction term for female-headed households and child stunting by provincial region was not statistically significant, suggesting that the relationship holds across the three culturally distinct regions. Conclusions for Practice Female empowerment was associated with lower rates of stunting among young children, and the results did not vary by provincial region. This suggests that women can play important roles as agents of change, even in areas where females have limited freedoms. Greater investments in public education and awareness campaigns to improve health literacy might have important spillover effects for child health and improve the success of existing public health interventions targeting childhood stunting.     

A detailed comparative analysis on the overall codon usage patterns in West Nile virus

West Nile virus (WNV) is a member of the family Flaviviridae and its genome consists of an 11-kb single-stranded, positive-sense RNA. WNV is maintained in an enzootic cycle between mosquitoes and birds, but can also infect and cause disease in horses and humans, which serve as incidental dead-end hosts. Understanding the extent and causes of biases in codon usage is essential to the comprehension of viral evolution. In this study, we performed a comprehensive analysis of 449 WNV strains, for which complete genome sequences are available. Effective number of codons (ENC) indicates that the overall codon usage among WNV strains is only slightly biased. Codon adaptation index (CAI) values found for WNV genes are different from the CAI values found for human genes. The relative synonymous codon usage among WNV strains isolated from birds, equines, humans and mosquitoes are roughly similar and are influenced by the relative dinucleotide frequencies. Taking together, the results of this work suggest that WNV genomic biases are the result of the evolution of genome composition, the need to escape the antiviral cell responses and a dynamic process of mutation and selection to re-adapt its codon usage to different environments.     

Culicoides Latreille (Diptera: Ceratopogonidae) taxonomy: Current challenges and future directions

Culicoides Latreille biting midges (Diptera: Ceratopogonidae) cause a significant biting nuisance to humans, livestock and equines, and are the biological vectors of a range of internationally important pathogens of both veterinary and medical importance. Despite their economic significance, the delimitation and identification of species and evolutionary relationships between species within this genus remains at best problematic. To date no phylogenetic study has attempted to validate the subgeneric classification of the genus and the monophyly of many of the subgenera remains doubtful. Many informal species groupings are also known to exist but few are adequately described, further complicating accurate identification. Recent contributions to Culicoides taxonomy at the species level have revealed a high correlation between morphological and molecular analyses although molecular analyses are revealing the existence of cryptic species. This review considers the methods for studying the systematics of Culicoides using both morphological and genetic techniques, with a view to understanding the factors limiting our current understanding of Culicoides biology and hence arbovirus epidemiology. In addition, we examine the global status of Culicoides identification, highlighting areas that are poorly addressed, including the potential implementation of emerging technologies.     

An improved Pythium insidiosum-vaccine formulation with enhanced immunotherapeutic properties in horses and dogs with pythiosis

The immunotherapeutic properties of a new Pythium insidiosum-vaccine formulation (PIV), was evaluated in 18 horses and 6 dogs with proven pythiosis from different enzootic areas in the United States. All injected horses but one responded with a weak (=29 mm, n=3), a mild (30-90 mm, n=7) or a strong (=100 mm, n=7) inflammatory reactions at the site of injection. Three equines with weak or negative reactions at the injection site were not cured. Seven equines with strong reactions at their injection sites, however, were cured. Six of the eight horses with mild reactions were also cured. The remaining two equines responded at first but both relapsed and finally died of their infections. The PIV cured only two of the six dogs used in this study. The new PIV formulation cured 72% of the equines (P=0.048) and 33% of the dogs with pythiosis. Dogs with chronic disease (greater than two months) did not responded to immunotherapy. The finding of eosinophils, mast cells, IgE and precipitin IgG during pythiosis suggested that a T helper 2 (Th2) subset is in place during this disease. In cured horses, the eosinophilic reaction was substituted by lymphocytes and mononuclear macrophages (Th1). This and previous studies strongly support the hypothesis that an immune-modulation from a Th2 to a Th1 subsets may be in part responsible for the PIV's curative properties.     

Identification of the encephalitis equine virus, Parana, Brazil

INTRODUCTION: In the period of 1996-1999 some virus associated with encephalitis have been reported in horses from different regions of Paraná State, Brazil. To identify the etiologic agent associated with this illness, mosquitoes and serum samples were collected in the endemic area. METHODS: The study area corresponded to four municipalities of Paraná State, Brazil. Mosquitoes were captured in Shannon trap and human bait. After identification, they were processed for virus isolation. Blood of equines were collected in the municipalities of Querência do Norte and Colorado. Antibodies to different Alphavirus and Flavivirus were analyzed by hemagglutination inhibition test. Specific seroneutralization reactions were performed in those sera with a positive reaction in the hemagglutination test. RESULTS: The mosquitoes genus collected were: Culex, Aedes, Mansonia, Coquillettidia, Psorophora, Sabethes, Wyeomyia, and Limatus. Even thought no virus was isolated, serologic analyses showed hemagglutinazing antibodies to Eastern equine encephalitis, Mucambo, Pixuna, Maguari, and St Luis encephalitis viruses. The neutralization test showed specific reaction to Eastern equine encephalitis virus in 12 tested sera. CONCLUSIONS: Species of mosquitoes that could be potential vectors of encephalitis, buniavirus, and other arboviruses of epidemiological importance were collected. It is believed that Eastern equine encephalitis virus affected the equines populations in the study regions because of the symptoms and antibodies for the virus in the sera detected in these equines.     

Venezuelan equine encephalitis vaccination survey in Arizona and New Mexico, 1972.

Field studies were conducted in 1972 to determine the immunization status of equines along the Mexico, Arizona, and New Mexico borders. Interviews with horse owners were conducted along roads selected at random in the counties of Santa Cruz and Yuma, Ariz., and in Dona Ana County, N. Mex. At least 450 horse owners in each county were asked about the vaccination status of their animals, and information was taken on 1,260 animals. Blood specimens were obtained from every third equine, regardless of stated vaccination status, and tested for the presence of Venezuelan equine encephalitis (VEE), western equine encephalomyelitis (WEE), and eastern equine encephalomyelitis (EEE) neutralization antibodies. Serum samples were collected from 446 equines in the 3-county area; only 227 (50.7 percent) had both a history of VEE vaccination in 1971 (including 20 vaccinated in 1972) and serum neutralization antibody against VEE. Of the remaining 220 with no detectable neutralization antibody to VEE, 197 (89.5 percent) had a history of VEE vaccination in 1971 (including 5 revaccinated in 1972), 14 (6.4 percent) had no history of vaccination, and 9 (4.1 percent) had an unknown vaccination status. Eighty-two percent (160 of 1971) of the equines with a history of VEE vaccination and presence of dectectable WEE or EEE antibodies, or both, had no detectable levels of VEE antibody. Therefore, the results of this study suggest that the presence of WEE or EEE antibodies, or both, may suppress the development of dectable vaccine-induced VEE antibody response in the equine. As a result of this investigation, the U.S. Department of Agriculture, as an added precaution, recommended the revaccination of equines in areas of the United States bordering Mexico.     

Malignant cancer and invasive placentation: A case for positive pleiotropy between endometrial and malignancy phenotypes

Cancer metastasis is an invasive process that involves the transplantation of cells into new environments. Since human placentation is also invasive, hypotheses about a relationship between invasive placentation in eutherian mammals and metastasis have been proposed. The relationship between metastatic cancer and invasive placentation is usually presented in terms of antagonistic pleiotropy. According to this hypothesis, evolution of invasive placentation also established the mechanisms for cancer metastasis. Here, in contrast, we argue that the secondary evolution of less invasive placentation in some mammalian lineages may have resulted in positive pleiotropic effects on cancer survival by lowering malignancy rates. These positive pleiotropic effects would manifest themselves as resistance to cancer cell invasion. To provide a preliminary test of this proposal, we re-analyze data from Priester and Mantel (Occurrence of tumors in domestic animals. Data from 12 United States and Canadian colleges of veterinary medicine. J Natl Cancer Inst 1971;47:1333-44) about malignancy rates in cows, horses, cats and dogs. From our analysis we found that equines and bovines, animals with less invasive placentation, have lower rates of metastatic cancer than felines and canines in skin and glandular epithelial cancers as well as connective tissue sarcomas. We conclude that a link between type of placentation and species-specific malignancy rates is more likely related to derived mechanisms that suppress invasion rather than different degrees of fetal placental aggressiveness.     

Vector incrimination studies and observations on species A and B of the taxon Anopheles culicifacies in Pakistan

Studies on anopheline mosquitoes in selected villages of Punjab Province, Pakistan have incriminated Anopheles culicifacies species A as the primary malaria vector. Although An. stephensi and An. subpictus showed higher immediate gut infection rates, estimations of relative abundance, age structure and survivorship, and observations of late gut and salivary gland infection rates suggested that neither species was a major vector in these villages. A survey of An. culicifacies populations detected the presence of both species A and B in several localities in Pakistan. Laboratory investigations demonstrated that both species are capable of supporting the extrinsic cycle of Plasmodium vivax. No recombination was observed between X-linked loci in species A/species B hybrid females; however, recombination was observed between loci in chromosome 2. Allelic tests between the X-linked white eye locus in species A and an X-linked white eye mutant in species B showed that the two loci are not allelic.     

Decreased access to medical care for girls in Punjab, India: the roles of age, religion, and distance.

Risk factors that increase the likelihood of discrimination against girls in India have not been well studied. In this study of hospitalized children in Punjab, India, girls were less likely to be in the newborn or infant age groups, to be of the Sikh religion, or to come from far away than were boys. These differences suggest that these factors are significant risk factors for denied access to medical care for girls living in Punjab, India.     

The measurement of liquid and solid digesta retention in ruminants, equines and rabbits given timothy (Phleum pratense) hay

1. Digesta passage and retention were measured in heifers, sheep, goats, equines and rabbits of varying body-weights when given timothy (Phleum pratense) hay. 2. Two passage markers were compared, cobalt (III) ethylene diamine tetraacetate (CoEDTA) and chromiummordanted timothy fibre for liquid and solid phase respectively. Both markers were injected into the rumen of the ruminants and into the caecum of the equines and rabbits. 3. In ruminants, two different sets of rate constants (k1 and k2) were derived from a two-pool model for marker passage, using a graphical approach and a computer-based non-linear least-squares curve-fitting technique. 4. Retention times, due to unidirectional flow through the gastrointestinal tract (transit time) and due to pool effects (mean retention time, MRT), were calculated. 5. Curve fitting was only successful for the excretion of liquids in ruminants. The two-pool model was not applicable to the passage of solids. 6. Apparent retention of liquid was always shorter than for solids in all species, except in rabbits. However, absorption of CoEDTA was too large in the rabbits to determine liquid retention accurately. Times for first appearance of the two markers were similar within animal groups. 7. MRT values were lowest in the rabbit, intermediate in equines and high in the ruminants. The MRT values (h) of solids and liquids respectively were: large heifers 65, 18; small heifers 48, 20; goats 41, 28; sheep 57, 26; equines 23, 18; rabbits 5.3, not determined. 8. Liquid retention seemed to decrease somewhat with increasing body-weight in the ruminants. Solids retention decreased with decreasing body-weight in the ruminants, but sheep had longer retention times than goats of similar size. Equines exhibited large individual variation in retention of the liquid or solid markers, seemingly unrelated to size. No effect of size was seen in the retention of solids in the rabbits.     

Environmental predictors of human West Nile virus infections, Colorado

To determine whether environmental surveillance of West Nile Virus-positive dead birds, mosquito pools, equines, and sentinel chickens helped predict human cases in metropolitan Denver, Colorado, during 2003, we analyzed human surveillance data and environmental data. Birds successfully predicted the highest proportion of human cases, followed by mosquito pools, and equines.     

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.     

Venezuelan equine encephalitis virus, southern Mexico

Equine epizootics of Venezuelan equine encephalitis (VEE) occurred in the southern Mexican states of Chiapas in 1993 and Oaxaca in 1996. To assess the impact of continuing circulation of VEE virus (VEEV) on human and animal populations, serologic and viral isolation studies were conducted in 2000 to 2001 in Chiapas State. Human serosurveys and risk analyses indicated that long-term endemic transmission of VEEV occurred among villages with seroprevalence levels of 18% to 75% and that medical personnel had a high risk for VEEV exposure. Seroprevalence in wild animals suggested cotton rats as possible reservoir hosts in the region. Virus isolations from sentinel animals and genetic characterizations of these strains indicated continuing circulation of a subtype IE genotype, which was isolated from equines during the recent VEE outbreaks. These data indicate long-term enzootic and endemic VEEV circulation in the region and continued risk for disease in equines and humans.     

Malaria vectors in the changing environment of the southern Punjab, Pakistan

The Pakistani Punjab experienced several devastating malaria epidemics during the twentieth century. Since the 1980s, however, malaria has been at a low ebb, while in other areas of Pakistan and neighbouring India malaria is on the increase. This raises the question of whether transmission in the Pakistani Punjab may have been influenced by a change in vector species abundance or composition, possibly induced by environmental changes. To investigate this question, routinely-collected government entomological data for the period 1970 to 1999 for the district of Bahawalnagar, in the Indus Basin irrigation system in the southern Punjab, was analysed. Our findings suggest that Anopheles stephensi has increased in prevalence and became more common than A. culicifacies during the 1980s. This shift in species dominance may be due to the large-scale ecological changes that have taken place in the Punjab, where irrigation-induced waterlogging of soil with related salinization has created an environment favourable for the more salt-tolerant A. stephensi. Some biotypes of A. stephensi are suspected of being less efficient vectors and, therefore, the shift in species dominance might have played a role in the reduced transmission in the Punjab, although further research is needed to investigate the effect of other transmission-influencing factors.