A large-scale study of a poultry trading network in Bangladesh: implications for control and surveillance of avian influenza viruses.

BACKGROUND: Since its first report in 2007, avian influenza (AI) has been endemic in Bangladesh. While live poultry marketing is widespread throughout the country and known to influence AI dissemination and persistence, trading patterns have not been described. The aim of this study is to assess poultry trading practices and features of the poultry trading networks which could promote AI spread, and their potential implications for disease control and surveillance. Data on poultry trading practices was collected from 849 poultry traders during a cross-sectional survey in 138 live bird markets (LBMs) across 17 different districts of Bangladesh. The quantity and origins of traded poultry were assessed for each poultry type in surveyed LBMs. The network of contacts between farms and LBMs resulting from commercial movements of live poultry was constructed to assess its connectivity and to identify the key premises influencing it. RESULTS: Poultry trading practices varied according to the size of the LBMs and to the type of poultry traded. Industrial broiler chickens, the most commonly traded poultry, were generally sold in LBMs close to their production areas, whereas ducks and backyard chickens were moved over longer distances, and their transport involved several intermediates. The poultry trading network composed of 445 nodes (73.2% were LBMs) was highly connected and disassortative. However, the removal of only 5.6% of the nodes (25 LBMs with the highest betweenness scores), reduced the network's connectedness, and the maximum size of output and input domains by more than 50%. CONCLUSIONS: Poultry types need to be discriminated in order to understand the way in which poultry trading networks are shaped, and the level of risk of disease spread that these networks may promote. Knowledge of the network structure could be used to target control and surveillance interventions to a small number of LBMs.

Incidence of contamination of live bird markets in Bangladesh with influenza A virus and subtypes H5, H7 and H9.

In the absence of robust active surveillance of avian influenza viruses (AIV) affecting poultry in South Asian countries, monitoring of live bird markets (LBMs) can be an alternative. In a longitudinal study of 32 LBM, five environments were sampled as follows: market floor, stall floor, slaughter area, poultry holding cage and water used for meat processing. Samples were taken monthly for 5 months, September 2013-January 2014. Incidence rates (IRs) of LBM contamination with AIV and its subtypes H5, H7 and H9 were assessed. In 10 of the LBM selected, biosecurity measures had been implemented through FAO interventions: the other 22 were non-intervened. Standard procedures were applied to detect AIV and three subtypes in pooled samples (1:5). An LBM was considered positive for AIV or a subtype if at least one of the pooled samples tested positive. The incidence rates of LBM contamination with AIV, H5, H7 and H9 were 0.194 (95% confidence interval (CI) 0.136-0.276), 0.031 (95% CI 0.013-0.075), 0 and 0.175 (95% CI 0.12-0.253) per LBM-month at risk, respectively. The log IR ratio between the FAO-intervened and non-intervened LBM for contamination with AIV was -0.329 (95% CI -1.052 to -0.394, p = .372), 0.598 (95% CI -1.593 to 2.789, p = .593) with subtype H5 and -0.500 (95% CI -1.249 to 0.248, p = .190) with subtype H9, indicating no significant difference. The results obtained suggest that both H5 and H9 were circulating in LBM in Bangladesh in the second half of 2013. The incidence of contamination with H9 was much higher than with H5.

Estimating the between-farm transmission rates for highly pathogenic avian influenza subtype H5N1 epidemics in Bangladesh between 2007 and 2013.

Highly Pathogenic Avian Influenza (HPAI) is classified by the World Organization for Animal Health as one of the notifiable diseases. Its occurrence is associated with severe socio-economic impacts and is also zoonotic. Bangladesh HPAI epidemic data for the period between 2007 and 2013 were obtained and split into epidemic waves based on the time lag between outbreaks. By assuming the number of newly infected farms to be binomially distributed, we fit a Generalized Linear Model to the data to estimate between-farm transmission rates (ß). These parameters are then used together with the calculated infectious periods to estimate the respective basic reproduction numbers (R0 ). The change in ß and R0 with time during the course of each epidemic wave was explored. Finally, sensitivity analyses of the effects of reducing the delay in detecting infection on a farm as well as extended infectiousness of a farm beyond the day of culling were assessed. The point estimates obtained for ß ranged from 0.08 (95% CI: 0.06-0.10) to 0.11 (95% CI: 0.08-0.20) per infectious farm per day while R0 ranged from 0.85 (95% CI: 0.77-1.02) to 0.96 (95% CI: 0.72-1.20). Sensitivity analyses reveal that the estimates are quite robust to changes in the assumptions about the day in reporting infection and extended infectiousness. In the analysis allowing for time-varying transmission parameters, the rising and declining phases observed in the epidemic data were synchronized with the moments when R0 was greater and less than one, respectively. From an epidemiological perspective, the consistency of these estimates and their magnitude (R0  ≈ 1) indicate that the effectiveness of the deployed control measures was largely invariant between epidemic waves and the trend of the time-varying R0 supports the hypothesis of sustained farm-to-farm transmission that is possibly initiated by a few unique introductions.

Biosecurity and Circulation of Influenza A (H5N1) Virus in Live-Bird Markets in Bangladesh, 2012.

Bangladesh has been considered as one of the five countries endemic with highly pathogenic avian influenza A subtype H5N1 (HPAI H5N1). Live-bird markets (LBMs) in south Asian countries are believed to play important roles in the transmission of HPAI H5N1 and others due to its central location as a hub of the poultry trading. Food and Agriculture Organization (FAO) of the United Nations has been promoting improved biosecurity in LBMs in Bangladesh. In 2012, by enrolling 32 large LBMs: 10 with FAO interventions and 22 without assistance, we assessed the virus circulation in the selected LBMs by applying standard procedures to investigate market floors, poultry stall floors, poultry-holding cases and slaughter areas and the overall biosecurity using a questionnaire-based survey. Relative risk (RR) was examined to compare the prevalence of HPAI H5N1 in the intervened and non-intervened LBMs. The measures practised in significantly more of the FAO-intervened LBMs included keeping of slaughter remnants in a closed container; decontamination of poultry vehicles at market place; prevention of crows' access to LBM, market/floor cleaning by market committee; wet cleaning; disinfection of floor/poultry stall after cleaning; and good supply of clean water at market (P < 0.05). Conversely, disposal of slaughter remnants elsewhere at market and dry cleaning were in operation in more of the FAO non-intervened LBMs (P < 0.05). The RR for HPAI H5N1 in the intervened and non-intervened LBMs was 1.1 (95% confidence interval 0.44-2.76), suggesting that the proportion positive of the virus in the two kinds of LBM did not vary significantly (P = 0.413). These observations suggest that the viruses are still maintained at the level of production in farms and circulating in LBMs in Bangladesh regardless of interventions, albeit at lower levels than in other endemic countries.

Risk factors for highly pathogenic avian influenza in commercial layer chicken farms in bangladesh during 2011.

A case-control study conducted during 2011 involved 90 randomly selected commercial layer farms infected with highly pathogenic avian influenza type A subtype H5N1 (HPAI) and 175 control farms randomly selected from within 5 km of infected farms. A questionnaire was designed to obtain information about potential risk factors for contracting HPAI and was administered to farm owners or managers. Logistic regression analyses were conducted to identify significant risk factors. A total of 20 of 43 risk factors for contracting HPAI were identified after univariable logistic regression analysis. A multivariable logistic regression model was derived by forward stepwise selection. Both unmatched and matched analyses were performed. The key risk factors identified were numbers of staff, frequency of veterinary visits, presence of village chickens roaming on the farm and staff trading birds. Aggregating these findings with those from other studies resulted in a list of 16 key risk factors identified in Bangladesh. Most of these related to biosecurity. It is considered feasible for Bangladesh to achieve a very low incidence of HPAI. Using the cumulative list of risk factors to enhance biosecurity pertaining to commercial farms would facilitate this objective.

Genetic diversity and phylogenetic analysis of highly pathogenic avian influenza (HPAI) H5N1 viruses circulating in Bangladesh from 2007-2011.

Highly pathogenic avian influenza (HPAI) H5N1 virus has been endemic in Bangladesh since its first isolation in February 2007. Phylogenetic analysis of the haemagglutinin (HA) gene of HPAI H5N1 viruses demonstrated that 25 Bangladeshi isolates including two human isolates from 2007-2011 along with some isolates from neighbouring Asian countries (India, Bhutan, Myanmar, Nepal, China and Vietnam) segregate into two distinct clades (2.2 and 2.3). There was clear evidence of introduction of clade 2.3.2 and 2.3.4 viruses in 2011 in addition to clade 2.2 viruses that had been in circulation in Bangladesh since 2007. The data clearly demonstrated the movement of H5N1 strains between Asian countries included in this study due to migration of wild birds and/or illegal movement of poultry across borders. Interestingly, the two human isolates were closely related to the clade 2.2 Bangladeshi chicken isolates indicating that they have originated from chickens. Furthermore, comparative amino acid sequence analysis revealed several substitutions (including 189R>K and 282I>V) in HA protein of some clade 2.2 Bangladeshi viruses including the human isolates, suggesting there was antigenic drift in clade 2.2.3 viruses that were circulating between 2008 and 2011. Overall, the data imply genetic diversity among circulating viruses and multiple introductions of H5N1 viruses with an increased risk of human infections in Bangladesh, and establishment of H5N1 virus in wild and domestic bird populations, which demands active surveillance.

Molecular characterization of a hyperinducible, surface membrane-anchored, class I nuclease of a trypanosomatid parasite.

The 3'-nucleotidase/nuclease (3'-NT/NU) is a surface enzyme unique to trypanosomatid parasites. These organisms lack the pathway for de novo purine biosynthesis and thus are entirely dependent upon their hosts to supply this nutrient for their survival, growth, and multiplication. The 3'-NT/NU is involved in the salvage of preformed purines via the hydrolysis of either 3'-nucleotides or nucleic acids. In Crithidia luciliae, this enzyme is highly inducible. For example, in these organisms purine starvation triggers an approximately 1000-fold up-expression of 3'-NT/NU activity. In the present study, we cloned and characterized a gene encoding this intriguing enzyme from C. luciliae (Cl). Sequence analysis showed that the Cl 3'-NT/NU deduced protein possessed five regions, which we defined here as being characteristic of members of the class I nuclease family. Further, we demonstrated that the Cl 3'-NT/NU-expressed protein possessed both 3'-nucleotidase and nuclease activities. Moreover, we showed that the dramatic up-expression of 3'-NT/NU activity in response to purine starvation of C. luciliae was concomitant with the approximately 100-fold elevation in steady-state mRNA specific for this gene. Finally, results of our nuclear run-on analyses demonstrated that such up-regulation in 3'-NT/NU enzyme activity was mediated at the posttranscriptional level.

A novel 4-kb interleukin-13 receptor alpha mRNA expressed in human B, T, and endothelial cells encoding an alternate type-II interleukin-4/interleukin-13 receptor.

A 4 kb human interleukin-13 receptor (IL-13R) chain cDNA was cloned from a B cell cDNA library using expressed sequence tags homologous to mouse IL-13R as probes. The deduced protein sequence shows a significant level of sequence identity with the IL-5R and the human IL-13R identified recently by expression cloning. The cytoplasmic region is very highly conserved between human and mouse homologs and contains a consensus binding motif for a signal transducer and activator of transcription. The cDNA encodes a protein binding IL-13 when expressed alone which participates in a receptor complex for both IL-4 and IL-13 when expressed in conjunction with the IL-4R alpha chain. Transcripts for this IL-13R chain could be detected in most tissues and organs studied and in T, B, endothelial cells, basophilic, immature mast cell, and monocytic cell lines. The pattern of expression is different from the other recently cloned IL-13R molecule, and correlates with sites where IL-4 and IL-13 signaling is known to occur. This novel receptor is therefore likely to be implicated in reactions involved in IgE responses, T helper 2 differentiation, adhesion of leukocytes to endothelium, and therefore in pathological phenomena such as allergy, atopy, and asthma.

Tumor necrosis factor alpha enhances the expression of the interleukin (IL)-4 receptor alpha-chain on endothelial cells increasing IL-4 or IL-13-induced Stat6 activation.

Functional receptors for interleukin (IL)-4 and IL-13 on endothelial cells consist of the 130-kDa IL-4 receptor alpha-chain (IL-4Ralpha) and a 65-75-kDa IL-13 binding subunit that are expressed in a ratio of about 1:3, respectively. The restricted number of IL-4Ralpha limits subunit heterodimerization and in turn receptor-mediated signaling. We report here, the effects of tumor necrosis factor alpha (TNF-alpha) on the expression of the receptor subunits for IL-4 and IL-13. By flow cytofluorometry and receptor-binding analysis of iodinated IL-4 and IL-13, stimulation with TNF-alpha-induced a 2-3-fold increase of the IL-4Ralpha expression. The up-regulation was also confirmed at the transcriptional level by reverse transcription-polymerase chain reaction. Radioligand cross-linking experiments revealed no change in the subunit composition of the TNF-alpha-induced receptor complex. Nevertheless, TNF-alpha stimulation led to increased activation of the IL-4-specific signal transducers and activators of transcription protein (Stat6) by IL-4 and IL-13. Thus, TNF-alpha corrects the subunit imbalance of the endothelial IL-4.IL-13 receptor complex thereby increasing receptor heterodimerization and in turn the signaling capability by IL-4 and IL-13.

Resistance to cerebral malaria in tumor necrosis factor-alpha/beta-deficient mice is associated with a reduction of intercellular adhesion molecule-1 up-regulation and T helper type 1 response.

Tumor necrosis factor (TNF) induced by Plasmodium berghei ANKA (PbA) infection was suggested to play an important role in the development of cerebral malaria (CM). We asked whether TNF-alpha/beta double-deficient mice, which have a complete disruption of the TNF-signaling pathways, are protected from CM and what might be the possible mechanisms of protection. PbA infection induces fatal CM in wild-type mice, which die within 5 to 8 days with severe neurological signs. In contrast, TNF-alpha/beta-deficient mice are completely resistant to PbA-induced CM. As PbA-induced up-regulation of endothelial intercellular adhesion molecule (ICAM)-1 expression as well as the systemic release of nitric oxide is found only in wild-type mice, TNF is apparently central for the recruitment of mononuclear cells and microvascular damage. Mononuclear cell adhesion to the endothelium, vascular leak and, perivascular hemorrhage are found only in the brain of wild-type mice. By contrast, the development of parasitemia and anemia is independent of TNF. Resistance to CM in TNF-alpha/beta-deficient mice is associated with reduced interferon-gamma and interleukin-12 expression in the brain, in the absence of increased T helper type 2 cytokines. In conclusion, TNF apparently is required for PbA-induced endothelial ICAM-1 up-regulation and subsequent microvascular pathology resulting in fatal CM. In the absence of TNF, ICAM-1 and nitric oxide up-regulation are reduced, and PbA infection fails to cause fatal CM.

Discrimination of Neospora caninum from Toxoplasma gondii and other apicomplexan parasites by hybridization and PCR.

Neospora caninum is a protozoan parasite which causes neurological problems in dogs and abortion in cattle. As N. caninum is difficult to distinguish morphologically from Toxoplasma gondii, we developed a molecular tool capable of discriminating between the two parasites. Genomic DNA was isolated from in vitro cultured N. caninum tachyzoites and cloned into a plasmid vector. Resulting colonies were subsequently screened by differential hybridization using N. caninum and T. gondii DNA. Two clones were characterized in detail: one clone, termed pNc5, was found to be specific for N. caninum whereas the second clone, pNc1, hybridized with DNA from both parasites. The sequence of pNc5 was determined and different oligonucleotide primers were designed for use in the polymerase chain reaction (PCR). A 944 bp fragment was specifically amplified from N. caninum DNA, but not from DNA extracted from T. gondii or different Sarcocystis species. Positive signals in PCR were obtained with as little as 100 pg parasite template DNA. In addition, dual PCR with primer pairs specific for N. caninum and T. gondii allowed the detection of either parasite in mixed samples.

Neospora caninum: specific oligonucleotide primers for the detection of brain "cyst" DNA of experimentally infected nude mice by the polymerase chain reaction (PCR).

Both tachyzoites and bradyzoites in tissue cysts of Neospora caninum are morphologically difficult to distinguish from those of other cyst-forming apicomplexan parasites such as Toxoplasma gondii. Thus, molecular tools may contribute to easy identification of the parasite. Based upon an N. caninum-specific DNA fragment that we have recently cloned, 5 sense (Np1, Np3, Np5, Np7, Np21) and 4 antisense (Np2, Np4, Np6, Np8) oligonucleotides were designed for a sensitive and specific polymerase chain reaction (PCR). Among 19 combinations of sense and antisense primers, the Np21/Np6, Np21/Np4, and Np7/Np6 primer pairs were found to generate specific single bands in the presence of at least 10 pg genomic parasite DNA as a template. The primer pair Np21/Np6 was able to detect a single tachyzoite in the background of DNA derived from 2 mg of brain tissue. In experimentally infected athymic ICR:nu/nu mice, N. caninum-DNA was detected consistently from brain tissue at days 13 and 18 after subcutaneous inoculation of tachyzoites. The presence or absence of the organisms in the cerebrum in either proliferative or cystic form was examined by immunohistological staining. The results indicate that PCR with the primer pair Np21/Np6 could provide an efficient tool for large-scale epidemiological studies using brain tissue obtained at necropsy.

[Diagnostic PCR in veterinary parasitology: tritrichomonosis, neosporosis/toxoplasmosis echinococcosis/cysticercosis]. / Diagnostiche PCR in der Veterinärparasitologie: Tritrichomonose, Neosporose/Toxoplasmose, Echinokokkose/Zystizerkose.

The PCR is now widely introduced as a diagnostic and epidemiological tool in veterinary and human parasitology. Certain parasitic infections are detected using PCR with much higher sensitivity compared to conventional methods, and novel molecular approaches are considered for the analysis of infectiological questions. A broad spectrum of parasites relevant for veterinary and human parasitology already can be diagnosed by PCR. In the present review article, PCR-based methods developped or applied at the Institute of Parasitology in Berne for the detection of protozoan infections (Tritrichomonosis, Neosporosis, Toxoplasmosis) and helminthic infections (Echinococcosis, Taeniosis) are summarized.

A procyclin-associated gene in Trypanosoma brucei encodes a polypeptide related to ESAG 6 and 7 proteins.

The procyclin genes of Trypanosoma brucei encode a family of glycoproteins expressed on the surface of procyclic forms of the parasite. These genes are present at different loci in tandem arrays of two or three copies depending on the strain. It has previously been shown that procyclin genes are transcribed from a promotor immediately upstream of the first procyclin gene in each cluster by an RNA polymerase that is resistant to high levels of alpha-amanitin. Here we show that additional genes, which we term procyclin-associated genes (PAGs), are located downstream of the procyclin genes and belong to the same alpha-amanitin-resistant polycistronic transcription units. A gene in the pro A locus, PAG 1, encodes a polypeptide that is related to the ESAG 6 and 7 proteins encoded in the VSG expression site. An unexpected feature of PAG 1 is that the major open reading frame of 405 amino acids only starts at position 1283 in the cDNA sequence and extends to the poly(A) tail. Sequences related to the 5' untranslated region of PAG 1 are also found downstream of procyclin genes in other loci, but the 3' coding region is unique to Pro A. This suggests that there are related PAGs which are coordinately transcribed with procyclin genes from different loci.

Suppression of mitogen- and antigen-induced lymphocyte proliferation by lanthanides.

Lanthanide ions (Ln3+) inhibited the proliferative response of human lymphocytes to various polyclonal mitogens and the 'purified protein derivative' (PPD) of the tuberculin antigen. Of the four Ln3+ ions tested lanthanum (La3+) was the strongest inhibitor; erbium (Er3+) and lutetium (Lu3+) were only weakly active, while samarium (Sm3+) had intermediate potency. At a concentration of 1 mM, La3+ almost completely inhibited the uptake of [3H]-thymidine by lymphocytes exposed to mitogenic agents. Trypan blue exclusion tests confirmed that the La3+ ions were not toxic. These findings may bear upon the reported anti-inflammatory properties of the lanthanides.