br Viral quasispecies profiles example data As an application of

Viral quasispecies profiles: example data
As an application of the indices described here, we present an example which consists in an analysis of three clinical samples of hepatitis C virus of different complexity whose origin has been described (Cubero et al., 2014) (Figs. 3 and 4) and which were used in a previous study of simulation with androgen receptor inhibitor indices (Gregori et al. 2014). They are three HCV NS3 amplicons, sequenced at a high depth and similar coverage (39006–40450 quality filtered reads). The three samples are identified as High, Mid and Low complexity populations. High corresponds to a high complexity HCV NS3 amplicon from a chronically infected patient. Mid corresponds to a different and more conserved NS3 amplicon of the same patient. Low is a rather homogeneous HCV NS3 amplicon from an acutely infected patient.
The sequence alignment of the High, Mid and Low complexity populations were used to calculate all the indices discussed in the text. The values calculated are compiled in Tables 1 and 2. Both HS, HGS, Mf, π, and Hill numbers with q≥1 provide the order High> Mid>Low, with the last two indices one order of magnitude apart between samples. The incidence measures (number of haplotypes, polymorphic sites, mutations, FAD, and Hill number with q=0) give High>Low>Mid (Tables 1 and 2).
The Montserrat plot which is a representation of the terms resulting in Mf, provides an example (Fig. 3): Low is a highly homogeneous quasispecies with a dominant haplotype representing 87% of the population, but with a long tail of low abundance single mutants, whereas Mid includes a lower number of mutations and haplotypes with three relatively abundant haplotypes with 1, 2 and 3 differences with respect to the dominant haplotype. The contribution of a high number of single mutants to the complexity of the Low population is similar to the contribution of a lower number of mutants at a higher abundance in the Mid population. The subpopulations that become apparent in the Montserrat plot may be used as a starting point to define a number of clusters in the partition analysis of quasispecies (PAQ), a non-hierarchical clustering method of quasispecies analysis developed by Baccam and colleagues (Baccam et al., 2001). PAQ may provide complementary information on viral population complexity, particularly when used with functional indices.
The Hill numbers profile is highly informative of the haplotype distribution structure, providing more complete information than any single abundance-based non-functional diversity index (Table 2 and Fig. 4 top). This profile (Fig. 4 top) shows the reversal, discussed above, between q=0 and q=1 due to the presence of a higher number of haplotypes, although at low abundances, in the Low respect with the Mid sample.
The polymorphic sites profile (Fig. 4 bottom) shows a higher genetic diversity of the Low sample with almost the double of polymorphic sites, whereas the mutation landscape of the Mid sample is dominated by just two relatively abundant mutations. Also, Fig. 4-bottom contributes to tone down the much higher complexity of the High sample with a polymorphic sites landscape dominated by few highly abundant mutations.
The matrix of haplotype pairwise distances may be visualized by specific profiles. The mutations profile by haplotypes (Suppl. Fig. 2) shows the number of haplotypes at each observed Hamming distance with respect to the dominant haplotype by a barplot. The pairwise distances profile (Suppl. Fig 1) shows the fraction of pairs of haplotypes at each observed Hamming distance. The functional attribute diversity profile (Suppl. Fig. 1) shows the contribution of each observed Hamming distance to this index. Finally, the nucleotide diversity profile (Suppl. Fig 2) shows the contribution of each observed Hamming distance between pairs of haplotypes to π. Instead of the Hamming distance any suitable genetic distance may be considered.

The preoperative planning was performed employing a

The preoperative planning was performed, employing a virtual plate by setting the position of the maxilla in Lefort type I osteotomy. The fragments of the maxilla bone and a plate were moved simultaneously and the displacement and angulation was calculated. The virtual model of Lefort titanium plate (Figure 2) was positioned after obtaining required movement on the virtual image using Freeform plus software. We did not use any computer-aided design and manufacturing drilling guide for the maxillary repositioning.
During the real operation, the positioning of real titanium plate in Lefort I osteotomy was done based on this preoperative planning; the maxilla was moved to the planned final position. To translate the virtual fixation plate accurately into a real plate, this distance from the geometric shape of the plate, anterior to nasal spine and teeth were measured using a Vernier caliper in the operating room. The intersection point (screw position) was measured using a compass. The vertical position was adjusted using 3D screw positions if there was a vertical dimensional change. The black arrows show screw positions before the bone was moved, and the white arrows show the screw positions after the bone was moved (Figure 3). The actual operation was carried out by employing the titanium plate fixed to the fragment of maxillary bone as planned. Informed written consent was obtained from the patient to report the case. The authors followed the Helsinki Declaration guidelines in this investigation.
The accuracy of the simulation results was analyzed using the 3D comparison function in Geomagic Qualify. Since the cranial p97 and zygomatic arch are not altered by the surgery, it was used for the superimposition. The differences between the virtual plans and the postoperative results are shown in Figure 4. The comparison of CT scans made before jaw surgery and immediately after surgery clearly showed the approach to be accurate and the error of the maxillary position was < 1 mm. Thus, it can be said that the preoperative planning employing a virtual plate may prove a reliable surgery.
Complex movements are performed in the three planes in bimaxillary surgery in particular. The main concern during orthognathic surgery is the transfer of preoperatively simulated p97 maxillary movements to the actual operation. Since the advent and incorporation of the new technology, the process has significantly changed the preoperative planning and simulation of surgery. We used 3D CT scan of the patient\’s face and titanium plate 3D scan to make preoperative planning.
Model surgery is now being eliminated from clinical routine practice and its place is being taken by 3D-virtual simulations performed on computer screens. Over the years, several articles have highlighted the importance of 3D planning in orthognathic surgery. In the present case, stressed community was easy to perform as it was planned in advance.
In the traditional plaster dental models only the patients\’ teeth are represented in three dimensions. The present 3D planning enables the craniofacial skeleton to be viewed at all times (while planning treatment and mobilizing osteotomized bone structures). Using the software option prediction of outcome, changes brought about in soft tissue can be visualized. In the present study, the 3D planning and simulated surgical scenario made the actual surgery easy and accurate; the error of maxillary position was < 1 mm. The ability of computerized 3D has led to what is now known as 3D planning. The software program enables the surgeon to interact with the 3D images and all data can be stored as computer files, which facilitates data management. All preoperative information can be reviewed repeatedly and easily. Although studies have been carried out on a number of software programs, not all of these can store preoperative data in one place and provide access to images, which serve to simulate surgery, draw osteotomy lines, and plan 3D treatment with the prediction of postoperative outcome. This was not possible with model surgery or 2D analysis since the postoperative results could only be predicted in the 3D treatment planning system. Working with 3D representations of the anatomic structures provides a more precise view of how the part of osteotomized bone is mobilized. When using 3D planning, all the necessary information is provided in images, which can be manipulated on a PC, whilst conventional planning makes it necessary to obtain data from different studies (radiographs, models and articulators, face bow, etc.) and to interpret the data before being able to develop a treatment plan. We also used the same technique to perform simulation surgery in the present case.

The surplus of the National fund

The surplus of the National fund continues to increase every year. From the time of the creation of the National fund, the major revenue was non-tax (sales of property and agricultural lands), but every year the tax-revenue share is becoming a larger portion of the total revenue of the National fund (Fig. 4).
Davis et al. (2001) suggest measuring the effectiveness of the oil revenue fund by its effect on the relationship between the government expenditure and resource export earnings. The data shows a possible positive correlation between Kazakhstan\’s government expenditure and export of oil (Fig. 5). Also, the correlation looks stronger before 2001, while from 2001 on (after creation of the National fund), government expenditure looks less correlated with oil exports.
Nevertheless, there is a strong relationship between the government\’s revenue and expenditure with oil prices (Fig. 6).
An economy\’s dependency on oil resources can be measured in two ways (Liuksila, Garcia, & Bassett, 1994): external dependency shows how a country relies on oil exports to obtain foreign exchange (the share of oil export to total export), while fiscal dependency on the other hand, measures how the public sector relies on oil revenues (the share of oil taxes to total fiscal revenue). Data which is necessary to calculate external dependency of Kazakhstan is available, but oil revenue of Kazakhstan, which is needed to calculate fiscal dependency, is not available. That is why payments of oil and gas companies to the government of Kazakhstan from National Reports on the implementation of the extractive industry transparency initiative in Kazakhstan 2005–2011 (available at were used as approximate data. These reports provide payments of selected oil, gas and mining companies. During the chemokine receptor antagonist 2005–2008 payments by oil and gas companies are not separated from payments by mining companies. From 2009 only tax payments to the budget from the oil and gas companies (excluding mining companies) were used. Since in 2005 only payments of 38 oil, gas and mining companies available, while in 2006–2011 payments of 103–170 oil, gas and mining companies are available, we calculated fiscal dependency over the period 2006–2011. Oil revenue is divided between the government budget and the National fund. That is why total fiscal revenue was calculated as a sum of government revenue and payments of oil companies which were deposited into the National fund. Employing these measurements and plotting the share of Kazakhstan\’s oil exports to its total exports against the share of oil revenue to total revenue shows that Kazakhstan has a high external oil dependency (Fig. 7). About 70 percent of export earning and 40 percent of the fiscal revenue can be definitively associated with the economic activities of the oil sector. For comparison, Fig. 8, based on the work of Liuksila et al. (1994) shows that Nigeria, Saudi Arabia and Venezuela, which are also oil-dependent countries, have over 60 percent external and fiscal dependency (Fig. 8).
As is the case for most oil-producing countries, Kazakhstan has benefited from the upward trend in oil prices that is largely attributable to the strong demand for energy products over the past few years. A simple test of correlation depicted in the graph below affirms the positive correlation between Kazakhstan\’s GDP and the world oil price and between Kazakhstan\’s GDP and the country\’s oil production (Figs. 9 and 10).
The portion of fund\’s revenue in Kazakhstan\’s total revenue is constantly increasing (Fig. 11).

Theoretical analysis

Empirical work


In 1997, Zbigniew Brzezinski who once served as President Jimmy Carter\’s National Security Advisor published his seminal book The Grand Chessboard: American Primacy and Its Geostrategic Imperatives with the goal of formulating a long-term strategy for US foreign policy in the post-Cold War period. The central argument of the book was that the US primacy in the world depended foremost on its success in maintaining political, economic and military dominance over the Eurasian supercontinent, which Brzezinski presumptuously depicted as “the chief geopolitical prize for America”. Such a bold assertion, however, was actually based on the self-confidence of the US policymakers of the period who boasted a strong political standing and powerful economy at home and an unequalled political, economic, military and cultural influence abroad in the immediate aftermath of the Cold War.

While the use of such a quantitative

While the use of such a quantitative technique may not fit well in a paper that claims to follow an ES approach to sustain its claims, I believe there is nothing that a priori prevents the mixing of methods to present more nuanced and more rounded conclusions (6 & Bellamy, 2011; Greener, 2011). As a BDS I of facts, while relying on mere qualitative analysis would prevent me from verifying how this convergence is played out in the UNGA, the mere quantitative aspect would prevent me from knowing on what norms there is convergence. This is why I endorse a multi-method analysis to disclose possible normative isonomy among Central Asian states in the UNGA. With respect to the last, quantitative part, it should be noted that contrary to Ferdinand (2013) I employ only one index of convergence, and not three. This is because this paper does not intend to be a quantitative analysis of voting convergence. Rather, one of its aims is to show a perhaps less precise but more rounded picture of normative convergence among Central Asian states, and therefore deems appropriate to be methodologically extensive and not intensive. In addition, I am aware that the use of these different methods does not provide a perfect picture of the normative convergence of Central Asian states within the UNGA, and some problems are still left out. These problems, however, will be dealt with in the conclusion, and will certainly (and hopefully) represent opportunities for further research.

Structure of the paper
The paper is structured as follows: in the first section, I will scrutinise and analyse the norms endorsed and supported in the official documents submitted to the UNGA by Central Asian states as a group. Therefore, in this section I will also focus on the “regionness” of Central Asia, meant as the ability to act on the world stage as a coherent group of states capable of acting in concert and presenting a common identity to the world. This section, in sum, will offer a collective overview on their normative convergence. In the second section, conversely, I will offer a discrete overview of their normative convergence. I will code declarations and speeches of each Central Asian state-representative to see what norms are adopted and embraced by singular leaders. As it can be expected, the hypotheses listed above will be validated if both the collective and the discrete overview will bring the same results. In the third section, I make a quantitative shift, and I will discuss the normative convergence of Central Asian states in voting sessions in the UNGA to verify if this convergence is also sustained at the decisional level. In the conclusions, I will discuss the findings of the research and the position of Central Asia in the global international society, as well as the value that an ES approach can add to the study of international politics in Central Asia.

Normative convergence in UN Documents submitted by Central Asian countries
The first examples of normative convergence and societal attitude among the five Central Asian states date back to the mid-1990s and the end of that period, when the Central Asian Union was still alive (1994–1998). Despite the already noted neglect of the wide literature on the matter, that period and that experience proved to be significant in terms of defining and sharpening the normative directions of Central Asia in world politics.
At the same time, the institution of Great Powers management is evident in the five states\’ assessment of the Afghan peaceful settlement: “[the sides] reaffirmed their readiness to continue their joint efforts to settle the situation in Afghanistan under the auspices of the United Nations, with the participation of neighbouring countries, the Russian Federation and the United States of America”. In addition, the regional identity of Central Asia was entrench and enhanced, signalling that all regional states adhered to those norms and principles:

apexbio calculator br Network supplying the hajjis

Network 3: supplying the hajjis – Afghan Turkmen and Uzbeks between Saudi Arabia and Yiwu
During four months of research in Yiwu in 2016, I met several traders from Afghanistan who told me of their work in Saudi Arabia. The majority of these traders identify themselves as being ethnically Uzbek and Turkmen, and as having been born in Afghanistan northern provinces, especially Balkh, Kunduz and Jowzjan. The family histories of these apexbio calculator however are also informed by migration and mobility. Most of them men I have spoken to have told me that their grandfathers came to what is currently Afghanistan from their homes and villages in Central Asia after the region was incorporated into the USSR in the 1920s. Trade was also important to the livelihoods of these families over the long term: before their migration to Afghanistan they were involved in animal husbandry and also the trade in pelts to Russia; after moving to Afghanistan they continued to be involved not merely in the trade of lamb pelt (karakul), but also of meat, carpets, and animal skins. Indeed, between the 1960s and 1980s some of Afghanistan\’s most well known traders came from families who had migrated to Afghanistan in the 1920s – they were respected for their production and distribution of carpets, the opening of transport companies that connected the cities of northern Afghanistan to Pakistan, Iran, and Turkey, as well as for establishing business and offices abroad, most especially in Hamburg and Istanbul.
This community\’s specific migration history has clearly affected the nature of their relations with Afghanistan. Unlike in the other Afghan commercial communities and networks that I have explored in this article, the majority of the men I have spoken to say they have never returned to Afghanistan after they left in the early 1980s. Those who theorise this say it is because they were already refugees in Afghanistan when they left, having been denied access to the Afghan national identity card (tazkira) until at some point in the 1960s. As a result, I am told, they felt little attachment to their homeland and instead sought to lead settled lives in the contexts to which they moved. Nevertheless, life in Saudi Arabia has also provided the opportunity for these traders and the communities to which they belong to explore their histories of connectedness. Saudi Arabia, like the UAE, operates they kafilah system (a Saudi system is required to be a partner of the business activities of a resident foreigner): some of my informants have told me that their kafilah are third generation descendants of 1920 emigres from the Emirate of Bukhara (see Balci, 2003). These people, they say, have ‘become Arab’ but are still more trustworthy partners than Saudi citizens without a shared Central Asian heritage.
As with the traders based in the former Soviet Union, merchants involved in the trade between China and the Middle East, are actively involved in the creation of diasporic community life in the settings in which they live and work. One such node of the affective and commercial life of this community is the old city in Jeddah, especially the Bukhari Street which has been home to communities of pilgrims, traders and migrants from Central Asia for over the past century. As we have already seen, another important neighbourhood in which commerce and familial life overlap for this community is the Zeytinburnu neighbourhood of Istanbul. Istanbul\’s Zeytinburnu neighbourhood has been home to Afghans of largely Uzbek and Turkmen background since the mid-1980s, but in the last four years the neighbourhood has become a vibrant site of Afghan sociability – not merely of Afghans seeking to travel to Europe. In the last four years, Zeytinburnu has become a vibrant site of Afghan sociability – not only of those coming to Europe about whom one reads a great deal in the news: many have moved here to trade and do business and have come from all over Eurasia. During visits to Istanbul that I made in July and October 2016, I met in the streets of Zeytinburnu traders that I have known in Tajikistan and Ukraine, as well as Yiwu and Kabul. There is a veritable assortment of Afghan restaurants and ‘watani’ handmade ice cream parlours to choose from. These are a real spectacle for Afghans and locals alike as the ice cream makers, who hail from northern Afghanistan, make their special delicacy by pounding cold cream in steel pots that are submerged in ice. Equally important are the community associations (vakif) that have been established by Jeddah-based traders in Istanbul. These are gathering spaces usually located in apartments or flats that have been officially registered with the Turkish state and that are used to hold community events, such as the funeral prayers for the deceased, wedding rituals, or meals organised as acts of charitable giving.

As discussed in the preceding

As discussed in the preceding paragraph, the bands associated with δS(NH3+) (1534cm−1), δ(H2O) (1594cm−1), ν(CO) (1644cm−1) and δA(NH3+) (1696cm−1) are relatively weak at atmospheric pressure. Except for ν(CO), the other three bands are not easily observed in the spectra of high pressures. However, the ν(CO) band splits at 9.9GPa and, the new band with high wavenumber disappears at 10.6GP as commented above. A possible interpretation of this behavior would be the coexistence of two phases at 9.9GPa, with a different frequency of the ν(CO) mode. The transition is complete at 10.6GPa, and hence only the mode corresponding to the high pressure structure remains.
Fig. 5 presents the pressure dependence of the Raman spectra in the spectral range 2850–3300cm−1 from 0.1MPa to 29.6GPa. From Ref. [17], the band observed originally at 2935cm−1 is associated with the symmetric stretching vibration of CH2, νS(CH2), the band at 2957cm−1 is associated with the stretching vibration of CH, ν(CH), and the band observed at atmospheric pressure at 2964cm−1 is associated with antisymmetric stretching of CH2, νA(CH2) [17]. A very weak band is also observed at 3115cm−1, which is assigned as antisymmetric stretching of NH3+, νA(NH3+). The pressure dependence of the wavenumber () of the three most intense bands is very different. Interestingly, between 0.1MPa and 9.3GPa d[νS(CH2)]/dP>d[ν(CH)]/dP, whereas at higher pressure, d[νS(CH2)]/dPglutamic referred to [12].
The pressure behavior of the νA(CH2) is impressive in MLA. The intensity of this band decreases with pressure up to 9GPa, when it increases at high pressure. In the spectrum taken at 6.6GPa its intensity is half of the intensity of the most intense bands. At 19GPa its intensity is twice that of the band associated with ν(CH). Above this pressure, the νA(CH2) loses intensity relative to the ν(CH) and, in the spectrum recorded at the highest pressure, all modes have a comparable intensity. However, the most impressive behavior of the νA(CH2) band is related to its high d/dP value, i.e., d/dP=12cm−1/GPa. As a consequence, (i) at P=29.6GPa, the wavenumber of the mode is =3310cm−1; (ii) the difference of wavenumbers between the bands of ν(CH) and νA(CH2) changes from 12cm−1 in the spectrum taken at 0.1MPa to approximately 140cm−1 in the spectrum recorded at 29.6GPa.
Fig. 6 presents the evolution of the Raman spectra of MLA in the spectral range 3300–3600cm−1 for pressure up to 29.6GPa. Three bands are observed around 3400cm−1 that, according to the Ref. [17], could be assigned as follows. At 3385cm−1 the band should be associated with the antisymmetric vibration of NH2, νA(NH2); at 3401cm−1, associated with symmetric stretching of H2O, νS(H2O) and at 3423cm−1, associated with the anti-symmetric stretching of H2O, νA(H2O). We observe that during compression the two bands shift to the lower wavenumber region of the spectrum. Such a behavior, as we will see in the Section 4, is characteristic of bands associated with modes involved in hydrogen bonds.

In a previous work some phase transitions have been evidenced below 1.3GPa [14]. We do not discuss these transitions because we were interested in the behavior of the material above 2.0GPa.
From the X-ray diffraction study at ambient pressure it is known that the chain of asparagine molecules is parallel to the c direction and that the adjacent chains are linked together through hydrogen bonds from the water molecules [13]. Additionally, there are hydrogen bonds between adjacent asparagine molecules without the participation of water such as the link between two of them along the a-axis. This picture suggests the phase transitions should affect both kinds of hydrogen bonds.

It should be remarked that the positions of

It should be remarked that the positions of peaks boundaries obtained with a Wth value very different from 1.6 leads to unsatisfactory results: in particular, as visible in Fig. 3 for the synthetic spectrum with 1% of white noise, for Wth=1 (Fig. 3a) and Wth=2.5 (Fig. 3b) the algorithm detects as peaks boundaries some spectral data which are clearly located inside spectral regions.
The red continuous lines in Fig. 2 are a rough estimate of background between two neighboring boundaries of spectral regions. Such an estimate can be improved by replacing the straight lines with polynomial functions, which are usually used for approximating the background underlying the Raman signal. Therefore, fitting processes through the n points corresponding to boundaries of spectral regions (n=5 in Fig. 2a, n=8 in Fig. 2b and n=7 in Fig. 2c) by using polynomial functions of different degrees, from 2 to n-2, have been carried out in order to evaluate the background for each spectral region. It is worth underlining that the background function of the whole spectrum has not been estimated by means of a single polynomial function, as usually reported in literature. In fact, we firstly estimated it for each single spectral region by using the polynomial function closest to the corresponding straight line. After such background estimation region by region, the background function for the whole spectrum is made by linking the results obtained for each spectral region, taking care to eventually minimize the small discontinuities at the AEG 3482 points by means of a smoothing procedure as described above in Section 2.3 point h. In fact, we found that such choice represents the best compromise i) to preserve a continuous shape (without mathematical discontinuities) of the background function, as it should be because it is related to fluorescence from cellular functional groups and stray light sources, and ii) to minimize underestimation or overestimation possibilities due to overlapping peaks. The obtained results for the background function are shown as blue continuous lines in Fig. 4. It is clearly visible that the obtained function estimates the simulated one (red dashed line) well for all the investigated synthetic spectra. On the contrary, single polynomial functions present a worse agreement with the simulated background. For example, a least squares fit over the whole 600⬜1800cm↙1 spectral region of the function shown as a blue continuous line in Fig. 4b to the simulated background is characterized by a sum of square residues equal to 7.3; instead, a similar fit of a single 2nd order, or 3rd order or 4th order polynomial function to the simulated background produces a larger sum of square residuals (equal to 17.2, 16.9 and 16.3, respectively).
After the background estimate, the Raman spectrum can be obtained by subtracting the background from the untreated spectrum: the obtained results in Fig. 4 for synthetic spectra show a good agreement between calculated (green continuous line) and generated (black dashed line) spectra.
We have also applied the developed method to cases where the true Raman spectra are not known. Measured Raman spectra of two different biological cell samples, consisting of in vitro cultured leukocyte and keratinocyte cells, are shown in Fig. 5a and b, respectively (black continuous line). The difference between the two spectra, apart from the relative intensity of some Raman peaks, is mainly the background signal, whose intensity with respect to that of the total signal is lower in Fig. 5a than in Fig. 5b. So, the former spectrum appears more noisy than the latter one. The estimate background, passing through the data (red points) delimiting the detected spectral regions, is also shown in the respective figures as blue continuous line. It has been obtained by fixing the Wf and Wth value at 6 and 1.6, respectively, for the lymphocyte sample and 4 and 1.6, respectively, for the keratinocyte sample. In both cases, the estimated background resembles a very broad Gaussian function, although it has been estimated by means of the linkage of different polynomial functions. Background eliminated spectra are shown as green continuous lines in Fig. 5. It is clearly evident that no artifacts (distortions, peaks erosion, spurious peaks) were introduced by our implemented method. In addition, these spectra show well resolved peaks for which the analysis of the spectral position, amplitude and broadening would be more easy to perform than for the measured spectra.

Although the conditions for egg development was near

Although the conditions for egg development was near optimal at 25 and 30°C, development was arrested at 35°C for many eggs at the UM/LM stage. Still, 4% reached L3 by day 31, which differs from earlier studies where no development was observed at this temperature (Roberts, 1937; Reid, 1960; Tongson, 1967). Despite the significant adverse effect of the temperature above 33°C on the development of the parasite eggs, results from a previous study on A. galli have shown that high temperatures (steam cleaning or hot water high pressure cleaning) did not prevent re-infection of the consecutive flock in the same barn (Höglund and Jansson, 2011).
Furthermore, short-term exposure (up to 24h) of fresh UM eggs to sub-zero temperatures (trial 3) had only minor effects on in ovo larval development following subsequent incubation at 25°C for 2 weeks. This is consistent with earlier findings (Levine, 1937; Roberts, 1937) and may suggest a strategy for cold tolerance in A. galli in accordance with other nematodes (Wharton and Allan, 1989), even if the mechanism in A. galli is still unknown. There is limited information on these aspects in A. galli (e.g. Ackert and Cauthen, 1931). Further work needs to be done to examine the effect of prolonged exposure to sub-zero temperatures and repeated freeze–thaw cycles on eggs in different stages of development.
To our knowledge, there are two previous papers on the moisture requirements for embryonation in A. galli eggs (McRae, 1935; Hansen et al., 1953). Together with earlier studies, our results suggest that approximately 85–90% RH or more is required for embryonation of A. galli eggs at the optimal temperature of 25°C (Table 3). Thus, our findings confirm the susceptibility of ascarid eggs to desiccation described in Gaasenbeek and Borgsteede (1998). Still, A. galli eggs might be able to develop at a temperature around 30°C and 70% atmospheric humidity in about 7 days. However, it glucokinase remains unknown whether A. galli can withstand repeated dehydration/rehydration cycles, and if the permeability of the eggshell is modified to control for water loss in response to desiccation. Although, knowledge about variations in the microclimate in poultry barns and in the outdoor environment is limited, it is believed that infective hotspots are generated in certain places where conditions are favourable.
Nematode eggs are in general considered to be highly resistant to different pH values due to the presence of a complex eggshell with an inner lipid layer that protects the developing embryo, particularly in ascarids (Bird and Bird, 1991). In the pH trial, the mean percentage of in ovo larval development was higher than 78% in the pH range 2.5–12.5, which confirms the high tolerance of A. galli eggs to extreme pH levels. Although neutral pH conditions are most likely to be found in the external environment, the eggs must survive gastric passage with a pH of 2.5–4.8 in the chicken proventriculus and gizzard (Denbow, 2000). Nevertheless, it has been shown that preservation of A. galli eggs in an acidic medium was detrimental to the establishment of infection in chickens (Lazdina and Grinberga, 1978). Similarly, the combination of high temperatures and aqueous ammonia inactivated A. suum eggs in toilet waste (Nordin et al., 2009).
It has been demonstrated previously for ascarids including H. gallinarum (Saunders et al., 2000) and A. suum (Gaasenbeek and Borgsteede, 1998) that periods of hypoxia will slow egg development. Similarly, in our study A. galli eggs were inhibited by anaerobic conditions, while most eggs remained viable and resumed development in response to oxygen. However, the long-term effects of hypoxia need to be further studied.
The broad-spectrum disinfectant chlorocresol (Interkokask, InterHygiene® GmbH) is one of the few products available today, claimed to be effective against ascarid eggs. Chlorocresol, as it was used in our study proved to be ovicidal in vitro even at a substandard concentration. However, for unknown reasons chlorocresol seemed not to be fully effective against A. galli eggs in poultry barns (Höglund and Jansson, 2011). Ascarid eggs are thick-shelled and have an outer sticky coat (Bird and Bird, 1991), by which they can adhere to surfaces and glucokinase equipment in poultry barns. In this way, A. galli eggs may escape exposure if disinfectant does not reach all cracks and crevices in poultry houses. Furthermore, the corrosive nature of chlorocresol prevents effective usage on equipment. Obviously, A. galli eggs are exposed to a set of more complex environmental condition in the field than in the laboratory.

br Acknowledgements This work was funded

This work was funded by the Wellcome Trust (WT) (grant no. 079445) through the Infectious Diseases of East African Livestock (IDEAL) project and the Institute of Tropical Medicine (ITM) through the University of Pretoria. Real time PCR was done at the ARC-OVI and the reverse line blots were done at the University of Pretoria. We thank Dr Olga Tosas and Dr. Magai Kaare who worked as the project manager before his untimely death in 2008. We pteryxin also appreciate the farmers who participated in the study, and the animal health assistants and laboratory technicians for their expert work in collecting and analysing the samples.

The southern cattle tick, Rhipicephalus (Boophilus) microplus (Canestrini), is one of the most damaging parasites of cattle in tropical and subtropical regions. Several chemical groups have been used for its control, including fipronil and lindane. However, in several countries, as a consequence of the extensive use of chemical acaricides, R. (B) microplus populations have developed resistance to the major pteryxin of acaricides, including fipronil (Castro-Janer et al., 2010a,b).
In Uruguay and Brazil, the resistance of R. (B.) microplus to various acaricides appeared sequentially in order of their introduction. Organochlorates (OC) were used at the beginning of the 1960s to control tick populations resistant to arsenic, but after three-four years, they were replaced when federal authorities authorized the use of organophosphates (OP) in Uruguay. Resistance of the southern cattle tick to OPs appeared at the end of the 1970s; thus, OPs were replaced by synthetic pyretroids (SP) at that time. At the end of the 1990s, amitraz and macrocyclic lactones began being used (Cardozo and Franchi, 1994). Initially, fipronil and fluazuron were not commonly used due to their expense. However, at the beginning of the 2000s, due to a reduction in their costs and rising problems of southern cattle tick resistance to other molecules, their use has increased. Currently, fluazuron is the only molecule without reports of resistance in Uruguay, and recently, resistance to fluazuron has been reported in Brazil (Reck et al., 2014).
Some classes of insecticides with different chemical structures may have the same target site. This can result in cross-resistance of the southern cattle tick to different classes of insecticides if the target site is modified (Casida, 2009).
Fipronil acts by blocking chloride ion channels that are regulated by gamma-aminobutyric acid (GABA) (GABA-Cl) present in the central neurons of the nervous system of insects (Cole et al., 1993; Durham et al., 2001; Bloomquist, 2003). Fipronil also blocks chloride ion channels that are activated by glutamate (Zhao et al., 2004; Narahashi et al., 2007). Cross-resistance between other GABA-Cl disrupters, such as cyclodienes (dieldrin and lindane) and phenilpyrazole insecticides (fipronil and others), has been demonstrated in other arthropods (Colliot et al., 1992; Cole et al., 1993, 1995; Bloomquist, 1994; Scott and ZhiMou, 1997; Brooke et al., 2000).
In both Brazil and Uruguay, in some cases, southern cattle tick resistance to fipronil has developed quickly after a few acaricide treatments (three to seven) (Castro-Janer et al., 2010a, 2010b). Thus, the objective of the present study was to determine the cross-resistance between fipronil and lindane in southern cattle ticks from Uruguay and Brazil.

Materials and methods

No mortality was observed among the controls during the bioassays. Table 1 shows the comparative toxicity of lindane based on reproductive parameters for the Mozo and RFSan strains. For the Mozo strain, the oviposition and the hatchability began to be affected at a lindane concentration of 62.5ppm. Concentrations lower than 250ppm had no negative effect on the surviving insects. The mortality was very high at 500 and 1000ppm (>80%). In contrast, reproductive parameters for the fipronil-resistant strain (RFSan) remained unchanged up to 500ppm. They began being affected at 1000ppm, when mortality was 53.3%.

It has been documented that the

It has been documented that the occurrence of ashworthiosis infection has gradually increased over recent years, especially in the European bison population (Demiaszkiewicz et al., 2013). Moreover, Osińska et al. (2010) conclude that the intensity of histopathological changes identified in the walls of the abomasa and duodena of infected animals is connected with the number of nematode specimens.
Until 2014, the presence of A. sidemi specimens in the abomasum and duodena of wildlife was confirmed during postmortem examination only. Moskwa et al. (2014) demonstrated that PCR can be used to detect DNA isolated from third stage infective larvae (L3) of A. sidemi to diagnose ashworthiosis in vivo in European bison. The susceptibility of sheep to A. sidemi has been confirmed in experimental infections (Kotrla et al., 1976). Based on the widespread occurrence of A. sidemi in wild ruminants in Poland, Dróżdż et al. (2003) discuss the possibility of the infection being transferred to domestic animals, such as cows and sheep, which may use the same pastures as wild animals. The current study applies PCR based on DNA isolated from the third stage infective larvae (L3) of A. sidemi to determine whether transmission of A. sidemi infection from wildlife to domestic animals, such as cattle, is possible.
Hence, the aim of the study was to examine cattle for the presence of A. sidemi in areas where the nematode parasite had only previously been reported in wildlife animals.

Materials and methods

The mixed population of larvae obtained after cattle-fecal sample incubation was examined by PCR to confirm the presence of L3 A. sidemi DNA. Positive results were observed in eight of the 250 cattle fecal samples tested. A. sidemi DNA was actually detected in DNA isolated from larval cultures derived from cyclooxygenase-2 inhibitors from cattle. Animals from two herds grazing in the buffer zone of Białowieża Forest area and two in the Strzałowo Forest District were found to be positive (Fig. 1). Agarose gel electrophoresis revealed the presence of a band of roughly 406bp after PCR amplification using the specific primers described earlier (Fig. 3). The specificity of the PCR reaction was confirmed by reference to the genomic DNA of adult O. circumcincta, H. contortus and C. oncophora, as well as muscle samples from bison (Fig. 4).
The obtained DNA sequence from A. sidemi L3 larvae was analyzed using BLAST software. A 99% match was found with the A. sidemi small subunit ribosomal RNA gene, partial sequence; internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and large subunit ribosomal RNA gene, partial sequence (accession numbers EF467325 and KF414629). The nucleotide sequence of the A. sidemi L3 larvae was deposited in the GenBank database under Accession No. KF414630.1.

Longitudinal studies by Dróżdż et al. (2003) and Demiaszkiewicz et al. (2009, 2013) confirmed that A. sidemi is widespread in wildlife ruminants in Poland. The results of a histopathological and parasitological study of bison during infection by A. sidemi incorporating a large number of parasites (77,630 specimens) confirmed that they have a significant impact on the health of European bison (Osińska et al., 2010; Demiaszkiewicz et al., 2013).
In most cases, a coproscopic or postmortem examination is required for a diagnosis of the presence of gastrointestinal nematodes. However, a multiplex PCR examination has also been created which can identify DNA from adult specimens of H. contortus, O. ostertagii, C. oncophora and A. sidemi: the procedure having been optimized on the basis of results from post-mortem examination by Ljungren and Goździk (2007, 2008). A later study by Moskwa et al. (2014) developed a simple PCR test based on DNA from the infective stage, which was found to be valuable in confirming or excluding A. sidemi infection in European bison in vivo. In the present study, genomic DNA from A. sidemi L3 larvae obtained from cattle fecal samples were amplified by simple PCR.