Sjaak De Wit

G D Deventer

Laboratory Fabri

Teun ; Gd

J J De Wit

W A J M Swart

T H F Fabri

FIGUES AND TABLES AT THE END OF THE MANUSCRIPT HERE, NOT FROM THE VERSION IN MANUSCRIPT CENTRAL The Figures can be put several to one page; I do not think they need to be too large The efficacy of infectious bronchitis virus vaccinations in the field: association between the α-IBV IgM response, protection and vaccine application parameters

Keywords: IBV, vaccination, field, IgM

The efficacy of infectious bronchitis virus vaccination in the field: association between the α-IBV IgM response, protection and vaccine application parameters

Sjaak de Wit, Teun Fabri, Wim

Disease vaccines [START_REF] Jackwood | Infectious bronchitis virus field vaccination coverage and persistence of Arkansastype viruses in commercial broilers[END_REF]. Many of these factors can have a negative effect on the efficacy of vaccination under field conditions. As many of these techniques and factors are developed or implemented for human comfort and are not necessarily favourable for the take of the vaccine virus, there is a need of a simple and cheap method to check the efficacy of IBV vaccination under field conditions. Routine determination of the level of protection by challenge is not practical or affordable. If a vaccination induces a humeral response, serology can be used for checking the vaccination efficacy by detection of a seroconversion using paired serum samples. This strategy is hampered for vaccination in the presence of maternally derived antibodies [START_REF] De Wit | Detection of infectious bronchitis virus[END_REF], the need for two samplings and the usually low serological response to mild vaccines [START_REF] De Wit | Sensitivity and specificity of serological tests for infectious bronchitis virus antibodies in broilers[END_REF]. An alternative strategy for serodiagnosing the take of an IBV vaccination would be to look for the presence of an IBV-specific IgM response. In contrast to the other immunoglobulin classes, like IgG and IgA, that are produced in response to IBV vaccination and infection, IBV-specific IgM response is short-lived [START_REF] Gillette | Avian infectious bronchitis: demonstration of serum IgG-and IgMneutralizing antibody by sucrose density-gradient centrifugation and mercaptoethanol reduction[END_REF][START_REF] Mockett | The detection of specific IgM to infectious bronchitis virus in chicken serum using an ELISA[END_REF][START_REF] Martins | A method for the rapid purification of serum IgM for the diagnosis of recent viral infections of chickens[END_REF][START_REF] Martins | IgM responses in chicken serum to live and inactivated infectious bronchitis virus vaccines[END_REF]Toro & Fernandez, 1994;[START_REF] De Wit | Detection of specific IgM antibodies to infectious bronchitis virus by an antibody-capture ELISA[END_REF]. Detection of IgM is therefore indicative of a recent infection or vaccination.

This paper reports the results of two field studies. In the first study, the efficacy of IBV vaccination in protecting against homologous challenge was compared with the results of the α-IBV IgM ELISA on sera that were collected at several intervals post vaccination of the same In a second field study, the IgM response to IBV vaccination was compared to detailed information of the vaccination process of 360 spray-vaccinated flocks of about 2-week-old broilers, layer pullets, broiler breeders and broiler grand parents. The information included parameters such as flock size, type of chicken, housing, age of the chicken, vaccine application route, vaccine used, dosage, water quantity, ventilation and light management, combination with other vaccines and temperature of the house and quality of water used. The aim was to identify factors that might be associated positively or negatively with the IgM response and thereby with the expected level of protection against homologous challenge under field conditions. This knowledge can be useful to improve the average efficacy of IBV vaccinations in the field.

Materials and methods

Field study 1, IgM IBV response and level of protection against IBV challenge. Study design and housing. At 13 days of age, 6 groups of 10 broilers were collected from 6 non-vaccinated broiler flocks and housed in separate negative pressure isolators at the Animal Health Service (AHS), The Netherlands. These birds were vaccinated (eye-nose drop) at 14 days of age with one dose of the same vaccine (combination of H120 and D274) as the remaining birds of these flocks housed in the field (Table 1). At 15 days of age, 10 birds from each of the flocks that had been vaccinated at the farm (spray or water application), were transported to the AHS and also housed in separate isolators. Blood samples were taken at 1 day before vaccination and 10 days after vaccination (d.p.v.) and stored at -20°C until testing. Non-vaccinated SPF broilers were used as control birds. Ciliostasis test. The level of protection was determined using the ciliostasis test on 5 tracheal rings per bird. The tracheas are placed in Hank's Minimum Essential Medium (HMEM) medium immediately after killing the chickens by an intravenous injection of 0.1 -0.2 ml of T61 (Intervet Nederland, Boxmeer, The Netherlands) and subsequent bleeding. Then, 5 rings (equally divided over the total length of the trachea) were cut and placed in medium at 37ºC. The level of ciliostasis was determined independently by two technicians between 1 and 4h after the birds were killed. The level of beating of the cilia for each ring was expressed as 0 (0% beating of cilia, total lack of protection), 1 (> 0 -25% beating), 2 (> 25 -50% still beating), 3 (> 50 -75% beating) or 4 (> 75 -100% beating). One bird could score between 0 and 20 (5 rings from each trachea: maximum score 4). An individual chick was recorded as protected against challenge if the ciliostasis score was 10 or more [START_REF] Cook | Breadth of protection of the respiratory tract provided by different live-attenuated infectious bronchitis vaccines against challenge with infectious bronchitis viruses of heterologous serotypes[END_REF]

IgM

Results

Field study 1, IgM IBV response and level of protection. The 6 groups of broilers that had been vaccinated by eye-drop at the laboratory showed at least 50% positive sera in the IgM ELISA at 10 d.p.v and a protection level against challenge between 89% and 100% (Table 1). The 6 groups of broilers that had been vaccinated at the farm showed on average 7% positives in the IgM ELISA at 10 d.p.v. and an average level of protection of 43% with a range from 0% to 86%.

Surprisingly, the differences in levels of protection between different houses on the three different farms were high. The differences in average protection of the birds of the different houses of the same farm were 51%, 76% and 36% for farms A, B and C respectively. According to the vaccinator the vaccination procedures had been the same on farm level, except for farm C, where there was a difference in dosage of the vaccine used, since House 1 received half a recommended dose of vaccine/bird.

Field study 2, IgM response and associations with vaccination parameters. Detailed information about the vaccination process was obtained from 360 flocks that had been vaccinated by the spray method (Table 2). The average percentage of responders was 46%. The results of the statistical analyses that were significant are summarized in Table 3. The average percentage of responders of the broilers (38%, 88 flocks) and of the layer pullets (46%, 178 flocks) were significantly lower than that of the grandparents (61%, 36 flocks). The average percentage of responders of the broiler breeders (52%, 58 flocks) was not significantly different from that of the other groups. Sixteen percent (95% confidence interval (CI): 8.3 -23.5%) of the broiler flocks had no detectable IgM response (all samples negative) compared to 0% of the grand parent flocks, 3% (95% CI 0 -8.1%) of the broiler breeders flocks and 7% (95% CI 3.5 -11.1%) for the pullet flocks (Figure 1). The percentage of broilers flocks without a detectable IgM response was significantly higher (P = 0.029) than the percentages of the other groups. For the broilers, 68% of the variation was explained by the variations in the parameters: flock size, ventilation, light and interval between vaccination and sampling time:

• Increasing flock sizes was correlated with decreasing IgM responses (P = 0.04) (Figure 2). Every extra 1000 birds resulted on average in a 1% lower IgM response (Table 3).

• Flocks that had been vaccinated with the ventilation turned off during spray vaccination showed a 15.5% higher IgM response (P=0.037) than the flocks that had been vaccinated with the ventilation system running (Table 3).

• Flocks that had been vaccinated with the lights turned on during spray vaccination showed a 41% higher IgM response (P=0.009) than the flocks that had been vaccinated

with the lights turned off (Table 3).

• A longer interval between vaccination and sampling resulted in an increasing IgM response (P = <0.0001) (Figure 3).

For the layer pullets, 44% of the variation was explained by the housing type and number of days between the previous IBV vaccination at day 1 and the vaccination around 14 days of age:

• Housing type. The average response for the birds housed in cages, an aviary system and on the floor covered with wood shavings was 31%, 48% and 53% respectively (difference cage-ground: P=0.010 and cage-aviary: P= 0.052, Table 3). There were considerable differences in average flock sizes for the housing types. The average flock size housed in the cages was 41,873 compared to 30,474 birds for the aviaries and 17,642 birds for the floor housing (Table 2). The parameters housing type and flock size were not independent from each other.

• A longer interval between the previous vaccination (Mass or Mass/D274) at day of hatch and the second heterologous 793B-type vaccination resulted in a higher IgM response (P=0.005, Figure 4). Every extra day between the vaccination at day 1 and the second vaccination (ranging from 8 to 19 days) resulted in a 2.5% higher IgM response (Table 3).

This was especially the case for the period of 8 to 13 days. Vaccinations from 13 days onwards resulted in comparable IgM responses. For the young replacement broiler breeders, the variation in the parameters was relatively small due to the number of vaccinators that were involved in the vaccinations of these flocks. 46% of the variation could be explained by:

• More days between previous vaccination (Mass or Mass/D274) at day of hatch and second heterologous 793B vaccination resulted in a lower IgM response (P=0.018) (Figure 5).

Every extra day between vaccination at day 1 and the second vaccination (ranging from 8 to 13 days) resulted in a 6.6% lower IgM response for each extra day after the second vaccination (Table 3).

• Higher temperatures of the water that was used to reconstitute and spray the vaccine (P = 0.021) resulted in lower IgM responses (Figure 6). Every degree (C) higher temperature (ranging from 6 to 18 °C) resulted in an average 3.2% lower IgM response after vaccination (Table 3).

For the young broiler grand parents, the variation in parameters was small. There were no significant correlations between the parameters investigated and the IgM results.

Discussion

This paper reports the results of two field studies. In a first, the efficacy of IBV vaccination against homologous challenge was compared with the results of the α-IBV IgM ELISA on sera that were collected at 10 d.p.v. The results showed that groups with at least 50% positive sera in the IgM ELISA at 10 d.p.v. had a protection of at least 89% against challenge. Groups of broilers with a low level of IgM positives showed an average protection rate of 43%, ranging from 0% to 86%. These results show that a higher percentage of positives in the IgM ELISA is an indication of a high efficacy of the vaccination. A low percentage of positives in the IgM ELISA should be interpreted as a warning, as the efficacy of the vaccination might be moderate, low or even nonexistent. It was concluded that the results of the IgM ELISA could be used as an indicator of the efficacy of the IBV vaccination in the second field study, which aimed to identify possible factors that might be associated positively or negatively with the IgM response and thereby with the expected level of protection against homologous challenge. This second study was performed to detect statistically significant associations between certain factors related to the vaccination procedure and management circumstances and the IgM response. However, it is important to realize that a significantly positive or negative association of a factor with a seroresponse, does not necessarily mean that it is a causal relationship. For example, if factor A causes a disease that results in manifestations B and C, there will be a statistical causal association between A and B, and between A and C. However, there will also be a statistical association between the two response variables, B and C, arising from their separate associations with A, but this is a noncausal association [START_REF] Martin | Veterinary Epidemiology, principles and methods[END_REF].

The average IgM responses of the broiler and layer pullet flocks was significantly lower than that of the broiler grandparents replacement flocks. It cannot be excluded that genetic aspects may play a role in this difference [START_REF] Cook | Investigations into resistance of chicken lines to infection with infectious bronchitis virus[END_REF][START_REF] Cook | Investigations into resistance of chicken lines to infection with infectious bronchitis virus[END_REF]Nakamura et al., 1991).

However, the difference in average results was highest between the broilers and the grandparents, which are most likely genetically more related to the broilers than the pullets (which were not significantly different). This makes it unlikely that genetic aspects play a major role. A major difference in the results between the type of chicken was the percentage of flocks with no detectable IgM response. Sixteen percent of the broiler flocks had no detectable IgM response compared to 0% of the broiler grandparent flocks, 3% of the broiler breeder flocks and 7% for the pullet flocks. Remarkably, this order is also the order of economical value of the birds, and this possibly had an influence on the attention and time that is invested in the vaccination procedure.

Another part of the explanation might be the effect of the difference in average flock sizes, a significant factor with the broilers, as the average flock sizes of broilers and pullets is higher than the relatively small flock size of broiler breeders and broiler grand parents. Another factor that might play a role, especially for the broilers, is the tendency to perform the vaccination of broilers in such a way that the vaccination reaction is not noticeable or is as mild as possible (Smith et al., 1985;[START_REF] Di Matteo | Morphologic observations on respiratory tracts of chickens after hatchery infectious bronchitis vaccination and formaldehyde fumigation[END_REF][START_REF] Matthijs | Ability of Massachusetts-type infectious bronchitis virus to increase colibacillosis susceptibility in commercial broilers: a comparison between vaccine and virulent field virus[END_REF][START_REF] Bijlenga | Development and use of the H strain of avian infectious bronchitis virus from the Netherlands as a vaccine: a review[END_REF]. Small disturbances in the performance of the broilers due to vaccination reactions in the short fattening period can already have a relevant economic impact, whereas this is hardly the case in the long living pullets, The statistical associations between the seroresponse and the ventilation and light management could be explained well with existing knowledge about IBV. Leaving the ventilation running during spray vaccination will reduce the vaccine virus concentration in the air because it is blown out of the house before it reaches the bird. The reason for the better response when the lights are on during the vaccination might be due to the fact that the eyes are open when the lights are turned on and closed when the light is turned off. It is well known that IBV vaccines applied onto the conjunctiva can be very effective [START_REF] Davelaar | Changes in the Harderian gland of the chicken following conjunctival and intranasal infection with infectious bronchitis virus in one-and 20-day-old chickens[END_REF]Survashe et al., 1979;Toro et al., 1996) and eye-drop application is generally considered to be the gold standard for application of live IBV vaccines.

The significant positive association between the interval between vaccination and sampling and the higher IgM response in the broiler flocks might well be caused by spreading of the vaccine virus after replication to some of the housemates. When only a part of the flock is effectively vaccinated, the vaccine may start to spread from bird to bird [START_REF] Matthijs | Transmissibility of infectious bronchitis virus H120 vaccine strain among broilers under experimental conditions[END_REF][START_REF] Jackwood | Infectious bronchitis virus field vaccination coverage and persistence of Arkansastype viruses in commercial broilers[END_REF]. This will result in a delayed IgM response at flock level.

The significantly lower IgM response detected in the layer pullets housed in cages compared to the birds on the floor and in aviary housing was not expected. The general idea was that birds in cages can more easily be vaccinated as they can not run away, although they can hide behind other birds in large cages. A potential reason for the lower response in the cages might be the usually big flock size (see negative correlation between flock size and IgM response of the broilers), but also the potentially fewer possibilities of spread of the vaccine from bird to bird compared to birds that are in contact with faeces in the case of suboptimal application of the vaccine.

A potential cause for the lower response in pullets when the vaccine was applied within 13 days of the first vaccination might be the recovery time needed by the tracheal epithelium after the first vaccination at day of hatch using a Mass strain or a combination of a Mass and a D274 strain (Winterfield & Fadly, 1972;[START_REF] Cook | Investigations into resistance of chicken lines to infection with infectious bronchitis virus[END_REF][START_REF] Di Matteo | Morphologic observations on respiratory tracts of chickens after hatchery infectious bronchitis vaccination and formaldehyde fumigation[END_REF][START_REF] Bijlenga | Development and use of the H strain of avian infectious bronchitis virus from the Netherlands as a vaccine: a review[END_REF]. Another explanation could be the findings of [START_REF] Davelaar | Influence of maternal antibodies on vaccination of chicks of different ages against infectious bronchitis[END_REF] who reported a decreased efficacy of H120 vaccination at 6 and 10 days of age, compared to vaccination at 1, 15 and 20 days of age. This decreased efficacy of a vaccination at 6 or 10 days was not confirmed in the work of [START_REF] Darbyshire | Humoral antibody response and assessment of protection following primary vaccination of chicks with maternally derived antibody against avian infectious bronchitis virus[END_REF], where they reported full protection against a M41 challenge at about 4 weeks post vaccination, using H120 at day 1, 7 and 14 in commercial layer pullets. However, these results were based on only 3 to 4 birds per group. The significant negative correlation between the length of the interval between the first, at hatch, and the second vaccination and the IgM response in the broiler breeders is in contrast with the results in the layers. A biologically plausible cause is hard to find. These results show that other, at present unknown factors are most likely interfering.

The extent of the influence of the temperature of the water used to reconstitute the vaccine (P = 0.021) was remarkable, but in line with the known susceptibility of IBV to higher temperatures [START_REF] Cavanagh | Infectious Bronchitis[END_REF]. Every degree (C) higher temperature (ranging from 6 to 20 °C) resulted in a 3.2% lower IgM response after the second vaccination.

As can be expected in these types of field study, not all results can easily be explained.

Both experimental and field studies have advantages and disadvantages. In experimental studies, the variables can often be well controlled, what makes it easier to draw conclusions from the results. Whether the results are relevant for the field is not always clear. Field trials are performed in the "real world", but the translation of the results can be very complicated due to the presence of many uncontrolled and unknown variables. However the results of these field studies provided information and knowledge that can be used for improving the average efficacy of IBV spray vaccination in the field. For this the IgM IBV ELISA is a helpful tool.

Acknowledgement

This research was supported by a grant from the National Board for Poultry and Eggs (PPE) of the Netherlands. We thank the contributing poultry farmers, veterinarians and integrations for their technical assistance to this work. 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 14 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13 13

F

  The efficacy of infectious bronchitis virus vaccination in the field: association between the α-IBV IgM response, protection and vaccine application parameters (IBV) is, in spite of vaccination, a major cause of respiratory problems in broilers and of poor egg production in breeders and layers in many parts of the world. Possible reasons of partial failure of the protection induced by vaccination are for example, heterologous challenges, immunosuppression, very short or long intervals between vaccination and challenge and last but not least, inadequate application of the vaccine. The mass application of IBV vaccines in the field is known for its many variations in a) application technique (eyedrop, course spray, drinking water, aerosol), b) quantity, quality and temperature of the water used to dilute the vaccine, c) dose, and d) the combination of different vaccines such as IBV with Newcastle

  predictive value of the IgM ELISA was determined for the efficacy of the vaccination.

  IBV ELISA. The ELISA was performed as described by De[START_REF] De Wit | Detection of specific IgM antibodies to infectious bronchitis virus by an antibody-capture ELISA[END_REF]. Briefly, a monoclonal antibody specific to chicken IgM was used as catching antibody and coated onto microtitre plates. Subsequent steps included the addition of test serum in duplicate (diluted 1:50 saline (PBS) pH 7.3 containing 0.65 M NaCl, 1 mM EDTA (Merck), 0.05% (v/v) Tween 80 and 0.5% (v/v) caseïne (Sigma)), IBV-M41 antigen or control-antigen (negative allantoic fluid), enzyme labelled anti-IBV monoclonal antibody (CVI-IBVS1-48.4), and enzyme substrate. Results were expressed as percentage positivity (PP%) compared to the results of the positive control (PC) sample. The PP% of a test sample (ts) was calculated as follows: PP% (ts) = {(OD ts (M41 antigen) -OD ts (neg. allantoic fluid)} x 100% OD PC (M41 antigen) -OD PC (neg. allantoic fluid) Sera with PP% results ≥ 14% were considered positive.Challenge. All birds were challenged by eye-drop with 10 4 median embryo infectious doses (EID 50 ) of the M41 strain of IBV in 0.1 ml of diluent per bird between 16 and 21 d.p.v. Nonvaccinated SPF broilers (AHS) were also challenged with M41 or with negative allantoic fluid at 49 days of age.

  grand parents. This tendency to vaccinate mildly, might logically result in a higher risk of inadequate take of the vaccine, resulting in low or no protection.

Figure 1 .

 1 Figure legends

Figure 2 .

 2 Figure 2. Association between the size of the broiler flocks and the average IgM response in each flock after IBV spray vaccination at approximately 2 weeks of age. Bar: 95% confidence interval of the estimated average percentage of IgM positive sera for each

Figure 3 . 20 Figure 4 .

 3204 Figure 3. Association between the number of days between vaccination and sampling and the average IgM response for broiler flocks after the IBV spray vaccination at approximately 2 weeks

Figure 5 .

 5 Figure 5. Association between the interval between the heterologous IBV vaccination in the

Figure 6 .

 6 Figure 6. Association between the temperature of the water was used to reconstitute and spray the vaccine and the average IgM response per broiler breeder flock after IBV spray vaccination at approximately 2 weeks of age. Bar: 95% confidence interval of the estimated average

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Overview of the IB IgM responses in each flock after IBV vaccination at approximately 2 weeks of age in 360 broiler, pullet, broiler breeder and broiler grandparent flocks. X-axis: 45 percentage of sera from a flock that was positive in the α-IBV IgM ELISA Y-Axis: percentage of flocks with a certain percentage of positive sera

Figure 4 .

 4 Figure 4. Association between the interval between the heterologous IBV vaccination in the hatchery and the spray vaccination under study and the average IgM response per layer pullet flock after IBV spray vaccination at approximately 2 weeks of age.Bar: 95% confidence interval of the estimated average percentage of IgM positive sera for each number of days 75 between the vaccination in the hatchery and the spray vaccination under study. Number on top of bar: number of flocks with that number of days between the vaccination in the hatchery and the spray vaccination under study.

Figure 5 .

 5 Figure 5. Association between the interval between the heterologous IBV vaccination in the

Figure 6 .

 6 Figure 6. Association between the temperature of the water was used to reconstitute and spray the vaccine and the average IgM response per broiler breeder flock after IBV spray vaccination

6 Field trial 2, IgM response and associations with vaccination parameters . Flocks

	. In the
	second field study, 360 flocks of broilers, pullets, rearing broiler breeders and broiler grand
	parents were vaccinated at about 14 days of age with an IBV vaccine by spray. All layer pullet,
	rearing broiler breeder, and broiler grand parent flocks had been vaccinated in the hatchery with a
	F o Massachusetts (Mass) strain vaccine or a Mass/D274 vaccine. In these flocks, the vaccine used at around 14 days of life was of the heterologous 793B (4/91,CR88) serotype (Table 2). The F o
	r vaccination schedule was the same in 78% of the broiler flocks. The other 22% of the flocks had r
	P e e r not been vaccinated in the hatchery and were vaccinated for the first time against IBV at P approximately 14 days of age with a Mass strain vaccine or a combination of a Mass and another IBV strain (D274 or Arkansas). Detailed information of the vaccination procedure and conditions in the house were recorded on an enquiry sheet. These data included for example the e e r
	R e v i e w type of bird, breed, age, flock size, vaccine used, dose, use of stabilizer, application method, amount of water allowed per bird, vaccination reaction, temperature of house, temperature of water to reconstitute and spray the vaccine, ventilation system, ventilation on/off, light on/off, other vaccinations (which, when), and season. At about ten days after the vaccination, blood was collected from 10 birds per house and tested for the presence of IgM against IBV by ELISA as described above. R e v i e w
	O n l y Statistical analysis. Analysis of Variance (UNIANOVA in SPSS 17.0) was used to analyze the data. This method has the prerequisites that the variance should be homogeneous within groups O n and that the residuals should be normally distributed. Because of lack of homogeneity in the variance the results of the broilers, pullets and broiler breeders had to be analysed separately. The l y
	ANOVAs were applied stepwise backwards starting with all potentially influential variables. At
	each step the least significant variable was removed and the analyses were repeated until all
	. For each group, a protection score (0 -remaining variables had a p-value of 0.05 or less. For the final model the normality of the
	100%) was calculated by the formula: residuals was tested.
	100% x (total of the individual scores) / (number of individuals x 20)
	7

Table 1 .

 1 Field study 1, IgM response and level of protection after IB vaccination at 14 days of age with a combined Massachusetts and D274 vaccine followed by a homologous Massachusetts M41 challenge

	Farm	House	Age at	Vaccinations applied on the farm	Vaccinations applied in the animal facilities at the AHS
			challenge	Vaccine	Dose a IgM 10	Protection	Vaccine application	Number	IgM 10	Protection
			with M41	application		d.p.v. b	score by	method	of doses	d.p.v.	score by
			(days)	method			ciliostasis				ciliostasis
	A	1	35	Spray	1	0	51	Eye-nose drop	1	77	100
		2	35	Spray	1	0	0	Eye-nose drop	1	50	99
	B	1	34	Drinking water	1	10	10	Eye-nose drop	1	70	96
		2	34	Drinking water	1	0	86	Eye-nose drop	1	60	97
	C	1	30	Drinking water	0.5	10	37	Eye-nose drop	1	77	91
		2	30	Drinking water	1	22	73	Eye-nose drop	1	90	89
	Average c					7%	43%			71%	95%
	SPF		49					eye-nose drop d			100
	broilers		49					eye-nose drop e			0

a dose of vaccine per bird b Percentage of IgM responders at 10 days post vaccination c Average of the 6 groups from farms A, B and C d Mock challenge with water applied by eye-nose drop e M41 challenge applied by eye-nose drop F o r P e e r R e v i e w O

n l y 25Table 2 .

 2 General information about the 360 flocks and the IBV spray vaccinations used for field study 2

	Parameter			Type of bird
			Layer pullets	Broilers	Broiler breeders	Broiler grandparents
	Number of flocks		178	88	58	36
	Housing system of flocks	cage	25%	-	-	-
		aviary	44%	-	-	-
		floor	30%	100	100	100
		unknown	1%	-	-	-
	Average flock size (range)		29,346 (4,700 -90,000)	25,238 (3,100-43,000)	10,042 (1,600 -22,700)	4,080 (540 -12,161)
	Ventilation during spray	Turned on	24%	39%	21%	14%
		Turned off	75%	61%	79%	72%
		unknown	1%			14
	Light during spray	Turned on	11%	7%	-	-
		Turned off	69%	92%	97%	100%
		unknown	20%	1%	3%	-
	IB vaccine used for revaccination	793B serotype	100%	78%	100%	100%
	at about 2 weeks of age	Mass serotype	-	14%
		Mass+ other serotype	-	8%
	Use of stabilizer with the vaccine	No	97%	20%	100%	100%
		Yes	3%	80%
	Average age in days at the IBV		13.2 (8 -28)	15.3 (10-21)	10.4 (8 -13)	12.8 (11 -14)
	spray vaccination (range)
	Average commercial vaccine		0.73 (0.55 -1.3)	0.86 (0.25 -1.29)	1.02 (0.94 -1.25)	1.05 (0.89 -1.51)
	dose per chicken (range)
	Average volume (ml) of spray		0.73 (0.20 -1.98)	0.56 (0.05-1.20)	0.65 (0.21 -1.75)	0.99 (0.75 -1.52)
	water per chicken (range)
	Average temperature (°C) of the		9.1 (4 -15)	11.8 (6 -25)	10.8 (6 -20)	10.6 (10 -13)
	water used to reconstitute and
	spray the vaccine (range)
	Average temperature (°C) in the		26.7 (22 -30)	25.9 (22 -30)	25.8 (23 -28)	25.0 (24 -27)
	house during the spray
	vaccination (range)
	Average interval in days between		10.0 (8 -14)	10.0 (8 -13)	10.3 (8 -14)	10.0 (9 -12)
	the IBV vaccination and blood
	sampling (range)

Table 3 .

 3 Statistical significant correlations between parameters of the IBV spray vaccination procedure and the average IgM response per flock in 360 flocks of approximately 2-week-old broilers, layer pullets, broiler breeders and broiler grand parents.

	Type of	Parameter	Category (number of flocks)	Average percentage of IgM	95% confidence interval of	P value (unless > 0.05)	Range of the parameter
	bird			positive sera per flock after	average percentage of IgM		tested in this study
				IBV spray vaccination	positive sera per flock
	All	Type of chicken	Broiler (88)	38%	32 to 45%	<0.001 (compared to the
						results for the broiler grand
						parents)
			Layer pullet (178)	46%	41 to 52%	0.004 (compared to the
						results for the broiler grand
						parents)
			Broiler breeder (58)	52%	46 to 60%
			Broiler grandparents (36)	61%	54 to 71%
	Broiler	Ventilation	On (34)	15.5% higher when the	1 -30%	0.037
			Off (54)	ventilation was turned off
				during the spray
		Light	Off (82)	41.1% higher when the	10.6 to 71.5%	0.009
			On (6)	light turned on during the
				spray
		Flock size		1.0% lower per 1000 extra	-2.0 to -0.1%	0.038	3.100 to 43.000
				birds
		Number of days between the		20.3%	10.6 to 29.9%	0.000	8 to 11 days
		IBV spray vaccination and
		blood sampling
	Layer	Housing type	Cage (44)	31%
	pullets		Floor (54)	53%		0.012 (compared with cage
						housing)
			Aviary (79)	48%		0.052 (compared with cage
						housing)
		Number of days between the		2.5% higher per extra day	0.8 to 4.3%	0.005	8 to 19 days
		vaccination in the hatchery and		between vaccination in the
		the spray vaccination under		hatchery and the spray
		study		vaccination
	Broiler	Number of days between the		6.6% lower per extra day	-12.1 to -1.2%	0.018	8 to 13 days
	breeders	heterologous vaccination in the		between vaccination in the
		hatchery and the spray		hatchery and the spray
		vaccination under study		vaccination
		Average temperature (°C) of		3.2% lower per extra	-5.9 to -0.1%	0.046	6 to 20°C
		the water used to reconstitute		degree (Celsius) of
		and spray the vaccine		temperature of the water

E-mail: cavanagh@metronet.co.uk URL: http://mc.manuscriptcentral.com/cavp Avian Pathology


T Hristova-Vasileva

V Vassilev

L Aljihmani

S Boycheva

Glass formation in the As 2 Se 3 -As 2 Te 3 -Sb 2 Te 3 system

Keywords: A. chalcogenides, B. synthesis, C. X-ray diffraction, D. thermodynamic properties

Chalcogenide glasses from the As 2 Se 3 -As 2 Te 3 -Sb 2 Te 3 system were synthesized for the first time. The glass-forming region was determined by X-ray diffraction and electron microscopic analyses.

The basic physicochemical parameters such as density (d), microhardness (HV) and temperatures of phase transformations (glass transition T g , crystallization T cr and melting T m ) were measured. Compactness and some thermomechanical characteristics such as volume (V h ) and formation energy (E h ) of micro-voids in the glassy network as well as the elasticity module (E) were calculated. The glass-forming ability was evaluated according to Hruby's criteria (K G ). The correlation between composition and properties of the (As 2 Se 3 ) x (As 2 Te 3 ) y (Sb 2 Te 3 ) z glasses was established and comprehensively discussed.

Introduction

A c c e p t e d m a n u s c r i p t 2

The chalcogenide glasses can be obtained by joined melting of elements from the chalcogenides group (S, Se and Te) with other elements, most often from the V th (As, Sb) and 4 th (Ge, Si) group of the Periodic table of elements. These glasses are unique due to their property to transmit light in the near and middle infra red parts of the spectra (from ~ 1 to > 18 µm [START_REF] Varshneya | Fundamentals of Inorganic Glasses[END_REF]) and by to this reason they are convenient for construction of IR-sensors, CO 2 lasers with application in the microsurgery, as well as for optical transport and storage media. These glasses are semiconductors with band gap values from 1 to 3 eV [START_REF] Varshneya | Fundamentals of Inorganic Glasses[END_REF][START_REF] Borisova | Halkogenidnie poluprovodnikovie stekla, Izd. Leningr. un-ta[END_REF].

The As and the Te form glasses in a narrow concentration area, which is in the range from 42 to 52 at. % Te (when cooled on air) and 54 at. % Te (when quenched in cold water) [START_REF] Eusner | Unusual electrical effects in As-Te semiconducting glasses[END_REF].

The addition of third component to the As-Te system leads to enlargement of the glass forming region [START_REF] Panus | Stekloobrazovanie v sisteme As-Ge-Te[END_REF][START_REF] Borisova | Stekla v sisteme Ag-As-Te[END_REF][START_REF] Dunaev | Stekla v sisteme As-Te-Ga[END_REF][START_REF] Dunaev | Stekla v sisteme As-Te-In[END_REF][START_REF] Savage | Glassformation and DSC data in the Ge-Te and As-Te memory glass systems[END_REF][START_REF] Hilton | New high temperature infrared transmitting glasses[END_REF][START_REF] Borisova | Stekloobrazovanie v sisteme As-Te-Tl[END_REF][START_REF] Panus | Stekloobrazovanie v sisteme As-Te-Cu[END_REF][START_REF] Vassilev | Glass formation in the As-Te-Sb system[END_REF].

The glasses from the As-Te-Sb system are perspective material for the integral microelectronics, but the glass forming region in this system is very narrow [START_REF] Vassilev | Glass formation in the As-Te-Sb system[END_REF], since there is no element in this system, which is a typical glass formator.

The introduction of Se will improve the glass formation in the As-Te-Sb system by widening the glass forming region and at the same time will not decrease

Experimental procedures

The glass forming region in the As 2 Se 3 -As 2 Te 3 -Sb 2 Te 3 system was outlined by the help of 27 compositions -Table 1. The characteristic temperatures of the glasses were defined by differential thermal analysis (DTA) using apparatus from the F.Paulic-J.Paulic-L.Erdey system of the company MOM-Hungary at heating rate of 10 °C min -1 , standard substanceα-Al 2 O 3 and accuracy of ± 5 °C.

The density (d) of the samples was measured with accuracy ± 5 % at air temperature of 25-30 °C by the hydrostatic method using toluene as immersion fluid.

The density of toluene is defined from the equation:

T 001 . 0 879 . 0 d - =
, where T is the temperature [°C], at which the measurement was made.

The microhardness (HV) was measured by the Vickers' method (a metallographic microscope MIM-7 with built in microhardnessmeter PMT-3 has been used at load of 10 g). The working values of HV are an average value of 20 measurements per sample; accuracy -± 5 %.

The elasticity module (E), the minimal volume of the micro-voids (V h ), the energy for their formation (E h ) and the Hruby's criteria (K G ) [START_REF] Wang | Formation and properties of chalcogenide glasses in the GeS 2 -Ga 2 S 3 -CdS system[END_REF] were calculated by the Eqs. ( 1) and ( 2) [START_REF] Aljihmani | Compositional trends of the physico-chemical properties in pseudoternary chalcogenide glasses[END_REF]:

HV 15 E = , HV T 04 . 5 V g h = , g h T 729 . 30 E = (1) cr m g cr G T T T T K - - = (2)
where T g , T cr and T m are the temperatures of glass transition, crystallization and melting.

A c c e p t e d m a n u s c r i p t

Results and discussion

The observations with non-armed eye and binocular magnifier (4 x ) show that the synthesized samples from the As 2 Se 3 -As 2 Te 3 -Sb 2 Te 3 system were dark colored, with strong luster and shell-like surface.

The results from the XRD show that some of the compositions (group a) are amorphous, without peaks on the diffractograms and with the characteristic X-ray amorphous plateau -these compositions form the glass forming region (Fig. 1). Other compositions (group b, Fig. 1b) show diffraction peaks with small intensity (compositions on the region's boundary). The diffractograms of the samples from group c show well expressed peaks. These samples are crystalline and get outside the region of glass formation (Fig. 1c).

The EMA investigations show smooth and homogenous surface for the samples from group a and for the compositions of group b small crystalline objects (microcrystals) appear. The surface of the samples from group c is rough, without the characteristic for the glassy phase regions. The temperature of glass transition T g varies in the range 125-165 °С (Table 2).

It decreases slightly with the increment of the As 2 Te 3 content and relatively more strongly with the addition of Sb 2 Te 3 . This course of the T g (m) and T g (z) dependencies is logical, since the glass forming ability decreases in the row As 2 S 3 → As 2 Se 3 → As 2 Te 3 , while the Sb 2 Te 3 is a crystalline substance and has never been obtained in glassy state.

Table 2. Thermal properties and Hruby's criteria of glassy phases from the (As 2 Se 3 ) x (As 2 Te 3 ) y (Sb 2 Te 3 ) z system.

In dependence of the composition, the crystallization temperature T cr varies in the range 177-229 °С (Table 2). On the thermogram of glass with composition (As 2 Se 3 ) 81 (As 2 Te 3 ) 9 (Sb 2 Te 3 ) 10 were not registered crystallization and melting effects which shows that this glass is stable and most probably As 2 Te 3 and Sb 2 Te 3 are built in the structure of the glass formator As 2 Se 3 in a way, which prevents the structural reorganizations leading to the amorphous-crystalline state transition. The lack of crystalline phase expels the existence of clearly presented endothermal melting effect.

The stability of the glass determines the presence of "diluted" protuberance in a direction corresponding to the endoeffect.

A c c e p t e d m a n u s c r i p t 8

For the compositions, corresponding to points 27, 7 and 12 (Table 1), situated near the glass formation border, two crystallization and melting temperatures were registered -Table 2. The presence of two exoeffects, corresponding to T cr , is logical since the probability of crystallization of more than one phase (solid solutions based on The melting temperature T m is in the range 320-377 °С (Table 2). The abovesaid for T cr is valid also for the T m , since T m (Sb 2 Te 3 ) > T m (As 2 Se 3 ) ≅ T m (As 2 Te 3 )

The Hruby's criteria K G gives qualitative idea for the glass forming ability of the alloys. K G changes in narrow limits (Table 2), as with the increment of the content of respectively18 [START_REF] Vassilev | Basic physicochemical properties of glasses in the GeSe 2 -Sb 2 Te 3 -CdSe system[END_REF], 150 [START_REF] Borisova | Halkogenidnie poluprovodnikovie stekla, Izd. Leningr. un-ta[END_REF] and 160 kgf/mm 2 [16] -Table 3. Due to the same reasons the HV depends slightly of the As 2 Te 3 concentration. .57 g/cm 3 [START_REF] Abrikosov | Poluprovodnikovie soedinenia, ih poluchenie i svojstva[END_REF]) -Table 3.

The compactness of the investigated glasses (Table 3) depends weakly of the Sb 2 Te 3 content (expressed by z at m=const), as well as of the As 2 Te 3 /As 2 Se 3 proportion (expressed by m=y/(x+y) at z=const). From the analysis of the compactness equation

[14] 1 F . d 1 x M d x M d x M d x M d x M d C i 1 n 1 i i i n 1 i i i i 1 n 1 i i i n 1 i n 1 i i i i i i - = - ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ ∑ ∑ = ⎥ ⎦ ⎤ ⎢ ⎣ ⎡ ∑ ⎭ ⎬ ⎫ ⎩ ⎨ ⎧ ∑ ∑ - = - = = - = = =
and rendering an account to the fact that The elasticity modulus follows the path of HV and changes within the limits 594-1270 kgf/mm 2 -Table 3.

The volumes of the micro-voids is in the range 8.0x10 -3 -17.7x10 -3 Å 3 (Table 3).

V h decreases when increasing the As 2 Se 3 content (z=const) and increases with the The energy for formation of micro-voids follows the path of Т g and varies from 3840 to 5000 kJ mol -1 (Table 3).

Conclusions

New chalcogenide glasses were synthesized and their glass forming region was outlined.

A number of thermal (temperatures of glass transition, crystallization and melting, Hruby's criteria), physicochemical (density, microhardness, compactness) and thermomechanical (elasticity module, micro-voids volume and energy for their formation) properties of the glassy phases were studied. A correlation between these properties and the composition of the glasses from the investigated system was established and a method for its explanation has been proposed.

A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t

A c c e p t e d m a n u s c r i p t 3 with

 3 significantly the mean molecule mass of the system. On the other hand the introduction of As and Sb in the form of their stable tellurides As 2 Te 3 and Sb 2 Te 3 , respectively, and of Se -in the form of As 2 Se 3 will keep this tendency. The transformation of the As-Te-Sb system, after substitution of a part of the tellurium with Se, into the As 2 Se 3 -As 2 Te 3 -Sb 2 Te 3 system presumes wider glass forming region (higher solubility of components near chemical and structural composition), higher crystallization stability, keeping to a certain degree the properties of the glasses from the elemental system, etc.The aim of the present work is the outlining of the glass forming region of the As 2 Se 3 -As 2 Te 3 -Sb 2 Te 3 system and the determination of the basic characteristics of the glasses in it.

A c c e p t e d m a n u s c r i p t 4 The

 4 obtained bulk samples were investigated through XRD (diffractometer TUR-M16 with CuK α -radiation and Ni-filter) and EMA (electronic microscope TEM Philips-3003).

Fig. 1 .

 1 Fig. 1. Typical diffractograms of samples from the As 2 Se 3 -As 2 Te 3 -Sb 2 Te 3 system: a)

A c c e p t e d m a n u s c r i p t 6 The

 6 region of glass formation in the As 2 Se 3 -As 2 Te 3 -Sb 2 Te 3 system (Fig.2), outlined on the basis of the results from the XRD and EMA analyses, is drawn to the As 2 Se 3 -rich region, lies fully on the As 2 Se 3 -As 2 Te 3 side (from 0 to 100 mol % As 2 Se 3 ) and partially on the As 2 Se 3 -Sb 2 Te 3 side (from 0 to 38 mol % Sb 2 Te 3 ). Glasses have not been obtained in the As 2 Te 3 -Sb 2 Te 3 system.

Fig. 2 .A c c e p t e d m a n u s c r i p t 7 Fig. 3 .

 273 Fig. 2. Glass forming region in the As 2 Se 3 -As 2 Te 3 -Sb 2 Te 3 system.

As 2

 2 Te 3 and Sb 2 Te 3 ) is fairly larger. T cr depends slightly of the As 2 Te 3 concentration, since the As 2 Se 3 and As 2 Te 3 are isomorphous, and much strongly from the concentration of Sb 2 Te 3 , as the addition of this component to As 2 Se 3 requires more complicated changes in the structure of the glass: partial substitution of As-with Sb-and of Se-with Te-atoms.

As 2

 2 Te 3 (z=const) and Sb 2 Te 3 (m=const) slightly decreases, which as it has been mentioned above, is due to the characteristics of the As 2 Te 3 and Sb 2 Te 3 .The microhardness is in the range of 40-85 kgf/mm 2 and logically decreases with the increase of the Sb 2 Te 3 content,

A c c e p t e d m a n u s c r i p t 9 The

 9 density of the glasses varies in the range 4.80-5.35 g/cm3 and logically increases with the increase of the As 2 Te 3 and Sb 2 Te 3 content (

increase of the Sb 2

 2 Te 3 content (m=const). This way of V h change from the composition is expected, since the exchange of As 2 Se 3 with As 2 Te 3 is connected to an exchange of Se-with Te-atoms, which ionic radii are -

A c c e p t e d m a n u s c r i p t 10

 10

Table 1 .

 1 Compositions used for outlining the glass forming region in the As 2 Se 3 -As 2 Te 3 -Sb 2 Te 3 system (m=y/(x+y))

	The starting compounds Me n Ch m (Me=As and Sb; Ch=Se and Te) and the
	glasses (4g) from the (As 2 Se 3 ) x (As 2 Te 3 ) y (Sb 2 Te 3 ) z system were synthesized by direct
	mono-temperature synthesis in sealed under residual pressure of 1.10 -3 Pa quartz
	ampoules. The initial materials used for the synthesis of the Me n Ch m compounds were
	Se and Te with purity 5N (Fluka) and As and Sb -3N5 (Merck). The synthesis
	characteristics (temperatures and duration of the isothermal steps, as well as heating rate
	between them) were in conformity to the physical and chemical features of the starting
	components, the intermediate and the final phases. At the maximum synthesis
	temperature of the glasses (700±10 °C) a vibration stirring of the smelter with duration
	2 hours was performed. The last was tempered at temperature of 650±10 °C and
	quenched in ice+water mixture with cooling rate 10-15 °C s -1 .

Table 3 .

 3 Physical and thermomechanical properties of glassy samples from the (As 2 Se 3 ) x (As 2 Te 3 ) y (Sb 2 Te 3 ) z system.

Table 1 .

 1 Compositions used for outlining the glass forming region in the As 2 Se 3 -As 2 Te 3 -Sb 2 Te 3 system (m=y/(x+y))

	Tables
	No	Composition, mol %		m	State
		As 2 Se 3	As 2 Te 3	Sb 2 Te 3
	1	9.5	85.5	5	0.9	Glassy + crystalline
	2	18	72	10	0.8	Glassy + crystalline
	3	25.5	59.5	15	0.7	Glassy + crystalline
	4	32	48	20	0.6	Crystalline
	5	37.5	37.5	25	0.5	Glassy + crystalline
	6	45	30	25	0.4	Glassy
	7	49	21	30	0.3	Glassy
	8	52	13	35	0.2	Crystalline
	9	58.5	6.5	35	0.1	Glassy
	10	72	8	20	0.1	Glassy
	11	56	24	20	0.3	Glassy
	12	40	40	20	0.5	Glassy
	13	81	9	10	0.1	Glassy
	14	63	27	10	0.3	Glassy
	15	36	54	10	0.6	Glassy
	16	45	45	10	0.5	Glassy
	17	32.8	49.2	18	0.6	Glassy + crystalline
	18	26.1	60.9	13	0.7	Glassy
	19	22.5	67.5	10	0.75	Glassy + crystalline
	20	19	76	5	0.8	Glassy
	21	9.7	87.3	3	0.9	Glassy
	22	4.85	92.15	3	0.95	Crystalline
	23	43.2	28.8	28	0.4	Crystalline
	24	46.9	20.1	33	0.3	Crystalline
	25	53.6	13.4	33	0.2	Glassy
	26	55.8	6.2	38	0.1	Crystalline
	27	63	7	30	0.1	Glassy

Table 2 .

 2 Thermal properties and Hruby's criteria of glassy phases from the (As 2 Se 3 ) x (As 2 Te 3 ) y (Sb 2 Te 3 ) z system.

	№	x	Composition y	z	m	T g	T cr	T m	∆T	K G
	13	81	9	10	0,1 165	-	-	-	-
	10	72	8	20	0,1 149	209	361	60 0,3947
	27	63	7	30	0,1 139 190/275 344/387 51 0,3566
	14	63	27	10	0,3 163	210	330	47 0.3916
	11	56	24	20	0,3 145	197	356	52 0,3270
	7	49	21	30	0,3 131 178/270 349/425 47 0,2748
	16	45	45	10	0,5 144	196	352	52 0,3333
	12	40	40	20	0,5 125 177/262 377/454 52 0,2600
	15	36	54	10	0,6 136	185	342	49 0,3121

Acknowledgement

The authors express their gratitude for the financial support for the performance of the present work to the Ministry of Education and Science -Bulgaria ("Scientific investigations" Fund -contract TN1503/05) and to the Science Research Sector of the University of Chemical Technology and Metallurgy (contract 10400).


Eavan G 1 Muldoon

Aideen Hogan

Dara Kilmartin

Cora 1 Mcnally

Bergin

Colm

Eavan Muldoon

Syphilis; consequences and implications in delayed diagnosis. Five cases of secondary syphilis presenting with ocular symptoms

Keywords: Syphilis, T. pallidum, ophthalmic, ocular, MSM

A Hogan-Ophthalmology care of patients and summary of ophthalmic presentation and findings D Kilmartin-Opthalmology care of patients and summary of ophthalmic presentation and findings C McNally-Medical care of patients and summary of cases C Bergin-Medical care of patients and review of article prior to submission

Key Messages

• Syphilis should be considered as an important treatable cause of uveitis. Untreated it could lead to irreversible vision loss. • Careful history may give clues to diagnosis; systemic symptoms are common prior to the onset of ocular syphilis. • HIV negative patients also present with ocular syphilis.

• Sexual health history should be incorporated into the evaluation of all patients.

Introduction

A large outbreak of syphilis was first described in Dublin in 2001 [START_REF] Hopkins | The great pretender returns to Dublin, Ireland[END_REF] The incidence of infection decreased until 2007 when a further increase was reported [START_REF] Muldoon | HIV and Syphilis Co-Infection; Emerging Trends Post Epidemic[END_REF] . Ocular manifestations of syphilis are uncommon. We present five cases diagnosed with ocular syphilis four of whom had a delay in their diagnosis. These patients presented over an eight-month period during which 118 diagnoses of early infectious syphilis were made in our centre. Four of the patients were men who have sex with men (MSM). Four patients were human immunodeficiency virus (HIV) negative.

Ocular syphilis has a broad spectrum of presentation. Posterior uveitis, retinal vascular involvement, placoid maculopathy, retinal detachment, involvement of the cornea, episcleritis and scleritis and interstitial keratitis presenting as open angle glaucoma have all been described [START_REF] Villanueva | Posterior uveitis in patients with positive serology for syphilis[END_REF][START_REF] Parc | Manifestations and treatment of ocular syphilis during an epidemic in France[END_REF] . Delay in the diagnosis of ocular syphilis can lead to irreversible visual loss due to optic nerve and retinal atrophy [START_REF] Aldave | Ocular syphilis[END_REF] .

Case reports

All five patients presented with decreased visual acuity. All patients were male, mean age 44.2 years (range 40-50) and four identified MSM as a risk for syphilis infection. Patient characteristics are outlined in table 1. All of the patients described systemic symptoms prior to onset of visual symptoms (table 1). One patient was known HIV positive; CD4 398 cells/mm 3 , virally suppressed on antiretroviral therapy. Three of five patients underwent lumbar puncture; all had a CSF pleocytosis. All patients received parenteral penicillin (either procaine penicillin and probenecid or benzylpenicillin for 14 days).

All of the HIV negative patients had a delay in the diagnosis of their syphilis. The time from onset of ocular symptoms to diagnosis ranged from six weeks to four months. The HIV positive patient had no delay in diagnosis. Three HIV negative patients attended for repeat HIV testing, all remained seronegative.

Discussion

Ocular manifestations of sexually acquired syphilis can occur at any stage of disease. The presentation of ocular involvement is varied, however the most common presentation of syphilis in the eye is uveitis. This can occur as early as six weeks after primary infection. When uveitis occurs in secondary syphilis it often follows the resolution of other signs and symptoms. The most common manifestations in patients with uveitis who presented to a New York clinic over a five-year period were granulomatous iridiocyclitis, non-granulomatous iridiocyclitis, panuveitis, posterior uveitis and keratouveitis [START_REF] Barile | Syphilis exposure in patients with uveitis[END_REF] . Anterior segment inflammation may be associated with a vitritis. Posterior uveitis is most commonly manifested as a chorioretinitis. As the clinical manifestations of syphilitic uveitis are non-specific, the work up for patients presenting with an unexplained uveitis should include testing for syphilis.

Sexual history may give a clue to the possible aetiology of a patient's uveitis, four of our patients were MSM and all reported episodes of unprotected sexual intercourse in the months preceding the onset of symptoms. These details were not elicited at initial presentation. Ocular involvement may be more severe in patients with HIV co-infection and is related to immune status [START_REF] Marra | Cerebrospinal fluid abnormalities in patients with syphilis: association with clinical and laboratory features[END_REF] . Syphilis is now more common than cytomegalovirus as a cause of uveitis among patients on anti-retroviral therapy [START_REF] Gaudio | Update on ocular syphilis[END_REF] .

As ocular syphilis is a manifestation of neurosyphilis, it should be treated with parenteral penicillin daily for a period of ten to seventeen days. Patients with reported penicillin sensitivity should be desensitized and treated with penicillin. The Jarish-Herxheimer (JH) reaction, a systemic reaction following antibiotic therapy can occur in the treatment of any stage of syphilis but is most common in secondary syphilis. It occurs one to two hours after administration of therapy. There are case reports of significant deterioration in symptoms or serious adverse events secondary to the JH reaction following therapy [START_REF] Pournaras | Bilateral giant retinal tear and retinal detachment in a young emmetropic man after Jarish-Herxheimer reaction in ocular syphilis[END_REF] . Steroid therapy prior to antibiotic therapy is recommended in patients with ophthalmic syphilis.

Ocular syphilis can be difficult to diagnose and if left untreated can lead to irreversible visual loss. If not considered during the evaluation of patients presenting with ocular symptoms of unclear aetiology the diagnosis may be missed. Four of our patients had a delay in the diagnosis of ocular syphilis after presenting with ophthalmic symptoms; all were HIV negative. Careful history taking regarding risks factors, history of possible chancrous lesions or systemic illness and or rash prior to the onset of visual symptoms may offer vital clues in making the diagnosis of this treatable disease.

Table 1 -Characteristics of patients presenting with ocular syphilis. * All non-treponemal tests were confirmed with T. pallidum particle agglutination (TPPA) ** Steroid therapy refers to steroids commenced prior to antibiotic therapy

 1

	Pt Risk	Preceding	Ophthalmic	Steroid	RPR* RPR	Response
			symptoms	findings	therapy	3	to therapy
					**	mth
	1	MSM	Malaise, fever,	Neuroretinitis &	Yes	1:256 1:16 Full
			lymphadenopathy	choroiditis			resolution
			& alopecia.				of
							symptoms
	2	MSM	Rash & general	Extensive	No	1:512 1:8	Full
			malaise.	chorioretinitis &			resolution
				disc oedema			of
							symptoms
	3	MSM	Rash.	Dense vitritis &	Yes	1:128 1:8	Severe
				choroiditis.			visual


László Sikos
email: sikos@dcs.uni-pannon.hu

Jiří Klemeš

Reliability, availability and maintenance optimisation of heat exchanger networks

Keywords: reliability, availability, maintenance, Heat Exchanger Network, RAMS, optimisation

A new methodology is proposed to use comprehensive up-to-date commercial software tools for Heat Exchanger Network (HEN) reliability modelling and optimisation. The idea behind this proposal is that to apply the combination of specific HEN optimisation and reliability software packages has several advantages over the commonly used approach. There is a variety of features that need to be taken into account to choose the right software tool. The HEN design has a significant impact on reliability issues and this should be considered. There are many related issues and features -the robustness, the type of welding, the increment of maximum mechanical resistance, the impact on manufacturing costs, reduction of lost opportunity costs caused by exchanger outages, troubleshooting of heating exchanger problems by operators etc. Fouling should be analysed as it has a significant impact on maintenance issues. Up to 30 % decrease of maintenance costs can be achieved annually by applying advanced reliability results and determining heat exchanger failure causes. These analyses include the investigation of failure causes, prediction of future probabilities of failures, cleaning planning and scheduling and the calculation of reliability and maintainability.

Introduction

A heat exchanger network is an important part of many processing and power generating plants. In most cases the HEN synthesis and design are assuming steadystate and non-variable operating conditions. In practice they can change and disturbances may occur.

Operational maintenance, availability and cost are some of the most important factors of HENs. All heat exchangers must be able to provide a specified heat transfer while maintaining a pressure drop across the exchanger. The propensity for fouling must be evaluated to assess the requirements for periodic cleaning. Fouling affects nearly every plant relying on heat exchangers for its operation. It is the accumulation of undesired solid material at the fluid or solid interface. Fouling introduces various costs, e.g. increased capital expenditure, and increased maintenance, loss of production, quality control problems, cleaning costs, additional hardware, and energy losses [START_REF] Kukulka | Transient Evaluation of Process Surfaces Used in Fouling Applications[END_REF].

Fouling of individual heat exchangers has been the subject of intensive research in recent decades. However, HEN fouling has even more issues. Maintenance considerations in the scheduling of continuous and batch plants have recently received increasing attention. Muller-Steinhagen [START_REF] Muller-Steinhagen | Handbook -Heat Exchanger Fouling -Mitigation and Cleaning Technologies[END_REF] proposed an integrated approach for developing alternative fouling mitigation strategies based on both experimental and modelling work. Georgiadis et al. considered the short term cleaning scheduling in special classes of HENs [START_REF] Georgiadis | Optimal cleaning policies in heat exchanger networks under rapid fouling[END_REF].

A wide variety of approaches are not limited to mathematical models and practical methodologies. The lifetime of heat exchangers varies with the application. A common practice to fouling mitigation is the implementation of Cleaning-In-Place (CIP) operations. Special heat exchangers exists which eliminate cleaning scheduling and maintenance activities by a self-cleaning mechanism. There are advanced materials, such as the hyper-duplex stainless steel, that designed and developed to increase operating performance and to extend service life in severely corrosive heat exchanger applications.

Reliability estimation is a useful tool to improve HEN design subject to uncertainties in the operating conditions. The efficiency of the technique has been proved by Tellez et al. [START_REF] Tellez | Heat exchanger network process modifications for controllability using design reliability theory[END_REF]. The analysis of the design constraints has been performed for different possible variations in the operating conditions.

Failure analysis contains Fault Tree Analysis (FTA). An FTA of a coolant supply to Heat Exchanger has been described as an example by Lazor in the reliability handbook of Ireson et al. [START_REF] Ireson | Handbook of reliability engineering and management[END_REF] Although several approaches and methodologies have been studied in the field of heat exchanger and HEN fouling, Reliability, Availability and Maintenance (RAM) issues of HENs should be further studied, especially for optimisation purposes. Scheduled and unexpected shutdowns should be differentiated. Maintenance times should be optimised. The characteristics of units should be considered. Another important issue is the mean time between maintenance and the reduction of efficiency to certain levels.

Present paper focuses on the possibilities of advanced software tools that support this field. The paper introduces a methodology for effective modelling and optimisation of HEN maintenance and reliability.

Problem statement

Market pressures drive management to achieve higher and higher levels of availability and reliability. However, the motivation to improve reliability is more complex than simply to reduce maintenance costs. There is a need to control reliability for best economic performance. In fact, least cost maintenance is not recommended when a plant strives to achieve high reliability. Maintenance costs are relatively low when compared with loss opportunity costs. HEN reliability issues can be effectively handled with RAMS software. The reliability issues of HENs deserve much more attention. Most designers mainly focus on HEN capital and operation cost optimisation only. The reliability issues of HENs should be properly analysed and improved by relevant software tools. All relevant features affecting availability, reliability and maintenance should be considered while modelling and optimising HENs. One of these factors is the interaction between heat exchangers in the HEN. The main task of optimisation is the appropriate scheduling of cleaning interventions of the individual exchangers in the HEN. It can be based on a priori knowledge of the time behaviour of the thermal resistance of fouling [START_REF] Markowski | Optimal cleaning schedule for heat exchangers in a heat exchanger network[END_REF]. Further tasks to be optimised are the operating costs of the HEN. The estimation of current and future failure probabilities are required to make the decision for equipment replacements performed at the right time to eliminate unnecessary shutdowns cased by unexpected faults. The simple cases, including series and parallel HENs, and the complex arrangements (block HENs) should be differentiated as their fouling factors are different. The detrimental effect of fouling can be reduced by adopting appropriate measures in HEN design [START_REF] Markowski | On-line cleaning schedule for heat exchangers in a heat exchanger network -The case of crude distillation unit[END_REF]. Important is the choice of local parameters and the HEN structure [START_REF] Brodowicz | Calculation of heat exchanger networks for limiting fouling effects in the petrochemical industry[END_REF].

Enhancing heat exchanger reliability has many advantages. Cost effectiveness can be increased; plant assets management can be improved. Component lifetime can be extended, and leakages can be detected prior to shutdown. Improved reliability results in reduced maintenance costs and a better economic performance.

RAM of Heat Exchanger Networks

Reliability is the probability that the HEN will perform satisfactorily for at least a given period of time when used under certain conditions [START_REF] Ireson | Handbook of reliability engineering and management[END_REF].

The availability of a Heat Exchanger Network represents the capability to manage heat and power streams continuously in a usual and regular way. The cause of failures varies, including mechanical failures, corrosion, fouling, and sealing problems.

Maintenance has an important role in plant design. Optimum maintenance planning is a key factor in modern HENs. Maintenance covers the activities undertaken to keep the HEN operational (or restore it to operational condition when a failure occurs).

A measure of ease and speed a system can be restored to operational status after a failure occurs can be expressed as a percentage. It is called maintainability, i.e. the probability of performing a successful repair action within a given time.

Analysing failures is an important way to determine availability and reliability issues of a system, including component failures, service failures, mechanical failures, control system failures (or malfunction), the failures to detect faults, changeover failures, lack of manpower, operator errors, and instrument failures.

The most important failure characteristics can be expressed by mean times. The widely used is the Mean Time Between Failures (MTBF), i.e. the reciprocal of failure rate. Further types of mean times are the Mean Time Before Maintenance Actions, the Mean Time Between Repairs, and the Mean Time To Failure.

Two types of fouling-induced effects should be identified in HENs [START_REF] Markowski | On-line cleaning schedule for heat exchangers in a heat exchanger network -The case of crude distillation unit[END_REF]: (i) Changes in outlet temperature of process streams caused by the thermal resistance of fouling in an exchanger;

(ii) Changes in inlet temperature of process streams caused by the thermal resistance of fouling in other exchangers ("antecedent exchangers" serving the same process streams). Some problems to be handled are the interaction between heat exchangers within the HEN and the optimisation of online cleaning.

Heat Exchanger Network system analyses

HEN analysis can be approached from various angles.

HEN design analysis

HEN design depends significantly on the types of the heat exchangers used. Several factors should be considered when selecting the heat exchanger type [START_REF] Peters | Plant Design and Economics for Chemical Engineers[END_REF].

Determining the list of system components is one of the first steps in availability and reliability analysis. The calculations require different kind of data of each component that build up the system. The component table could be useful to describe these data, because the majority of parts have different characteristics. The next step is to build up a system tree (either as a drawn tree structure or as a table).

Failure analysis

Failure Analysis (FA) or Root Cause Analysis (RCA) is a detailed examination of failed items to determine the root cause of failure and to improve product reliability. It helps with developing tests focused on problematic failure modes and with selecting better materials and/or designs and processes, and with implementing appropriate design changes to make products or processes more robust [START_REF]Design for Reliability: Overview of the Process and Applicable Techniques, In: Reliability EDGE[END_REF]. FA is strongly supported by reliability software tools.

RAM analysis

Availability analysis identifies the items that can affect system operation. It can be extended by considering the combination of maintainability and reliability data. System Reliability Analysis with Reliability Block Diagrams (RBDs) can be used for testing systems via component reliability and overall system reliability. Weak points can be identified. Different designs can be compared. Maintainability analysis provides data for the optimisation of maintenance and repair actions. These analyses can be performed in all major RAMS software packages.

Comprehensive software for modelling

The significance of the latest approaches of modelling the behaviour of the heat transfer surface by using the fouling resistance factor increased recently. As a result of intensive research, some of the classical theories are either being validated or modified to suit current design and optimisation techniques.

Various types of modern software packages are used for modelling the different issues of HENs. Fouling mitigation strategies can be effectively modelled by Fuzzy-Logic Expert Systems (FLES) and Computational Fluid Dynamics (CFD) software. General MILP solvers can be used for optimisation of processes.

However, another kind of software can be used for modelling and optimising the reliability related issues of HENs: Reliability, Availability, Maintainability and Safety (RAMS) software. The proper choice of RAMS software depends on many factors (e.g. the place of application, the amount of provided data, calculation precision). Availability can be treated together with reliability and life cycle cost modelling as they are influenced by each other. Some examples of RAMS packages are BlockSim and Weibull++ (ReliaSoft [START_REF]BlockSim and Weibull++ software[END_REF]), Reliability Studio (Relex Software Corp. [START_REF]Relex Reliability Studio[END_REF]), Item ToolKit (ITEM Software [13]). Most of them can be tested as a trial version for free. A more detailed description was introduced by Sikos and Klemeš [START_REF] Sikos | Reliability and maintenance software in energy generation and saving[END_REF]. There are specific CAD approaches as well, e.g. process design software tools applied for capacity increase and advance of distillation units, performing predictions of fouling layer structure or improving design [START_REF] Thernesz | Applying process design software for capacity increase and revamp of distillation units, Clean Technologies and Environmental Policy[END_REF]. Prototype software 'FiltraDynaSim' for the analysis of the fouling layer properties in microfiltration was introduced by Tung et al. [START_REF] Tung | Analysis and prediction of fouling layer structure in microfiltration[END_REF] It can be used both to design a membrane filtration system and to predict or monitor its performance during plant operation. Nordman and Berntsson presented a graphical method to determine heaters and coolers for retrofit in HENs [START_REF] Nordman | Use of advanced composite curves for assessing costeffective HEN retrofit I: Theory and concepts[END_REF]. They used advanced composite curves to describe the potential amount of heat that could be saved. They can serve as a monitoring tool to identify heat recovery targets and show the order of units to be targeted for rearrangement. Bertolini et al. determined an algorithm that allows a methodical, automatic approach for management of failure data in oil refineries [START_REF] Bertolini | Development of riskbased inspection and maintenance procedures for an oil refinery[END_REF]. It seems to be a significant potential to make substantial improvements in task organisation and decision-making processes. The aim of the Risk-Based Inspection and Maintenance (RBI&M) method proposed is to solve two important problems of reliability of refineries: (i) The personnel available for the analysis of critical items and events is limited and it is not able to assess in detail all the events occurred; (ii) Once defined the critical level of an item or event, reliability department has to decide the best maintenance actions and work orders to carry out. Kim et al. introduced a systematic methodology for designing wastewater and heat exchange networks [START_REF] Kim | A simultaneous optimization approach for the design of wastewater and heat exchange networks based on cost estimation[END_REF]. Based on cost estimation, networks for water and heat exchange were optimised simultaneously. Their proposal is a useful guideline for wastewater and heat exchanger network design with greater cost efficiency and environmental performance. The method employed a specific strategy to address mixed integer non-linear programming (MINLP) formulations. The mathematical formulation was based on water-pinch analysis and an expanded transhipment model. Bulasara et al. [START_REF] Bulasara | Revamp study of crude distillation unit heat exchanger network: Energy integration potential of delayed coking unit free hot streams[END_REF] addressed a revamp study of the crude distillation unit (CDU) heat exchanger network (HEN) of a typical refinery with and without the consideration of the free hot streams available in the delayed coking unit (DCU). Two sub-cases of revamp study have been considered: (a) Installation of new heat exchangers for the entire network; (b) Reutilisation of existing heat exchangers. They demonstrated the relevance of the consideration of DCU free hot streams in CDU revamp and retrofit design choices.

A suggested methodology

The proposed methodology suggests a combination of specific HEN optimisation tools with reliability software packages to improve the effectiveness of reliability optimisation in heat exchanger networks. A structure to conduct reliability, availability and maintainability analyses is presented as well.

The reliability analysis of HENs can be performed manually or using reliability software packages. The main advantages of using RAMS software in HEN reliability modelling and optimisation are: RAMS software packages allow total system approaches in analyses of individual system components. This contributes to the optimisation of design targets for plants to improve equipment selection, replacement, maintenance, as well as to increase system reliability.

The reliability program

When making the decision which tube bundles to replace, reliability groups should look at the reliability engineering principles and applying their results. The heat exchanger reliability could be supported by specification of the scope of the reliability analysis, which should contain:

• The total number of failures

• MTBF • Fluid velocity
• Years of service • The current age of heat exchangers. From them further requirements are recommended to investigate operation classes, date of installation, date of last replacement, tube age factor table, remaining life factor table, production criticality factor, service conditions, bundle replacement costs, and other considerations (e.g. corrosion mechanism).

Data requirement

Numerous data required to perform reliability and maintainability calculations. They can be classified in three groups.

First, general characteristics needed, including the general description of the plant, HEN usage, the period of continuous HEN operation, system structure, HEN arrangement. Data collection time interval need to be settled for further experiments. Features to be considered include but not limited to the list of equipments, units, flow streams of the HEN, the heat duty, the heat transfer area, heat transfer coefficients, shell and tube passes, the number of tubes, and design ΔT. The main groups of candidate variables are: operational (tube/shell inlet temperatures, flowrates, and integral flows), maintenance (peak efficiency), scheduling (crude/condensate in blend), and predicates (shell/tube side outlet temperatures).

Availability and reliability calculations are in the second group. They require failure history, durations of failures and shutdowns. Three classes of heat exchanger operation modes should be differentiated both for forced (unplanned) shutdowns (e.g. tube leakage) and scheduled maintenance actions. Equipments of Class A require total unit shutdown. In Class B, production cutback is required by a certain percentage. Class C equipments have no impact on system output (run-to-fail).

The optimisation of maintenance actions need variables depending on the system to be analysed. In a crude oil plant, for example, the ones that affect HE fouling required, including operational variables (inlet and outlet variables, flow rates of the crude, fluid temperatures, wall temperature of tubes, tube side and shell side flow rates), maintenance variables (e.g., peak efficiency), scheduling variables (crude/condensate in blend), as well as crude property variables.

Analyses can be more easily understood if a figure of the HEN is provided. Process data are required as well, including inlet and outlet, mass flowrate (both for shell side and tube side), the exchanged heat, and the heat transfer surface.

System reliability analysis with RAMS software

System reliability analysis can be divided into parts. Firstly, the required reliability approach should be decided. The steps for conducting reliability analysis of HENs are:

(i) Data collection: gather specific bundle failure history.

(ii) Conduct Weibull analyses on the failure data.

(iii) Failure probability calculations to estimate current probability of failure. During this step, the various operation classes of heat exchangers should be differentiated. Both the maintenance activities that ensue after a failure and the failure mechanisms at work affect the way of conducting the Weibull analyses of heat exchangers. For tube failures caused by process corrosion, failures are expected to reveal a wearout pattern, represented by β > 1. If the Weibull analysis results in β < 1, the heat exchanger is failing due to lack of quality or some other process that occurred during manufacturing.

To control heat exchanger reliability, the probability of failure should be accurately predicted. Estimating tube bundle reliability should account for the age of each individual tube that failed. Each tube lifetime is estimated from the installation date of the bundle to the failure date, at which time the failed tube is plugged.

Case study

The proposed methodology is suitable for optimising maintenance issues of HENs. A HEN of a petroleum refinery plant has been selected to demonstrate its capabilities.

The MOL (Magyar Olaj-és Gázipari Nyrt.) plant is located in Százhalombatta, Hungary. The refinery has a conventional Crude Preheat Train which consists of a set of shell and tube heat exchangers. Fouling of this unit reduces heat input (Fig. 1). Based on experience, energy consumption is 10-20 % higher due to fouling. Fouling level is mainly depends on the properties of the crude blends being processed, the operating temperature, and flow conditions. Online cleaning is not applied.

The analysed subsystem contains 36 heat exchangers and 2 desalters (Fig. 2). The estimations and calculations (Table 1) were based on the failure data of the last two years (2007/2008) provided by the company (Table 2).

There were no large unexpected faults in 2007. Because of the small problems occurred during in various parts of the year, the Mean Time Between Failures increased through 2008, when two considerable faults happened. However, the downtimes caused by these events were not as much as can be expected, thank to the spares available at the right time [START_REF]MOL refinery, Százhalombatta, Hungary -Personal consultation[END_REF].

The measured failure data were inserted into the spreadsheets of Relex Reliability Studio. The fault tree diagram was generated based on the failure tables (Fig. 3.). The advantage of this approach is the inclusion of their impacts on each other.

Weibull analysis and the estimation of future probability of failure were performed by Reliasoft Weibull++.

There were four main types of system events in the analysed subsystem: normal events that do not have impact on downtimes, scheduled outages such as repairing or cleaning, unexpected faults and outages caused by special orders. The most important events of them are the unexpected faults that should be reduced or even eliminated. Predictions performed through failure analysis are efficient to make suggestions to this field.

Mean Time To Repair has become approximately four times larger in 2008 than it was previous year, i.e. the repair rate decreased to one forth. The further result is the most important from our point of view: maintainability has fallen significantly within the examined period. The time needed to restore the system to fully operational status varies from a few minutes to some days due to the complexity of repair and maintenance tasks. In 2007, maintainability reached 100% within less than 1 day. In 2008, the plant achieved the same level of maintainability within a maximum of four days (Table 3).

The software tools proved that the examined subsystem of the plant runs in a nearly optimal way. However, small corrections could improve RAM issues, depending on probable investments.

Inherent availability was 99.539 % in 2007 and 98.530 % in 2008. The plant had high system reliability, too (97.307 % and 96.003 % in the last two years).

Class B operating mode is preferred in the plant so the production efficiency becomes lower continuously. Total shutdowns are not required for maintenance actions (eliminating Class A mode). However, production cutback does not performed by a fixed threshold. Instead, cleaning is done during major scheduled outages as preventive maintenance.

The plant has high maintainability. Maintenance, repair and replacement actions are well organised and scheduled. Many actions do not cause more than a few minutes outage due to spares. Failures are rare and mainly caused by heat exchanger breakdowns, leakages, problems in the steam production, and the burnout of the tube furnace. Maintainability cannot be increased significantly by simply increasing the frequency of cleaning interventions. However, the failure causes mentioned above are strongly related to availability and reliability and thus they can be optimised via total system approach supported by reliability software.

A longer period should provide more detailed picture because of the large intervals between main scheduled cleanings of up to four years. However, based on the performed study, some recommendations still can be added and the weak points highlighted. In the worst cases, fouling reduced the diameter of tubes by one third. At the same time, heat transfer coefficient went down because of fouling. A threshold should be applied to prevent very high fouling levels, especially where rapid fouling occurs. Scheduling of major outages should be improved while still considering costs. Supervisions should be done more frequently. However, total shutdown was not necessary and total downtimes were not significantly longer than the sum of the short downtimes in 2007. The most sensitive points in the system from the point of maintainability are the frequency of cleaning to avoid the effects of fouling, the failed heat exchangers and the efficiency of repairs (e.g. the repair of tube furnace burn control).

Conclusions

The proposed software modelling and optimisation methodology provides a way to reduce future losses in HENs and reach these aims through optimum maintenance. Several new approaches have been reviewed. The proposed methodology focuses on HEN maintenance through the influence of availability and reliability rather than the optimisation of cleaning schedules only. It has been shown that the failure analysis is capable to predict heat exchanger bundle replacement times, leading to significant savings. All major failure types are taken into account, including heat exchanger breakdowns, fouling, and leakages. Units of HENs are classified through operating classes and the most specific maintenance characteristics of fouling. This can reduce or even eliminate the need for total unit shutdowns required for cleaning. Reliability culture of plants or subsystems can be developed via total system approach, considering antecedent exchangers, HEN structure, local parameters, spares, and cost. The case study demonstrated that the RAMS software approach is capable to find the weak points in HEN maintenance, and emphasize the small corrections that could improve these issues towards optimality.

Figure Captions

  (i.) Prediction of replacement times can eliminate costs via failure analysis. Performing Weibull analysis on the failure data yields an estimation of MTBF and provides parameters that can be used to estimate future probability of failure. Based on Weibull parameters, it can be determined if exchangers in the HEN will probably survive until the next scheduled outage. If not, what is more cost effective: to replace now or to wait for the next scheduled outage? The answer if it is worth to wait until the next outage is delivered. (ii.) Estimation of current probability failure with failure probability calculator. (iii.) Realization of the establishment of new reliability culture at the plant. (iv.) Improvement of system reliability. (v.) Reduction and optimisation of maintenance costs. (vi.) Reduction of loss opportunity costs. (vii.) Optimisation of the balance between maintenance costs and loss opportunity costs via cost benefit analysis. (viii.) Control over equipment reliability. (ix.) Cohesive modelling modules. System tree, FMEA

  HENs can be analysed by calculating the reliabilities of individual series and parallel sections and combining them in the appropriate manner. (xii.) A wide variety of distributions. The standard definition of maintainability M(t) = 1 -e -μt can be easily expanded to different distributions. In case of the Weibull distribution, for example, MTTR should be modified from 1the t values are the times to failure, ' σ t = standard deviation of the natural logarithms of the times to failure.

Fig. 1 .Fig. 2 .Fig. 3 .

 123 Fig. 1. Effects of HE fouling in the plant Fig. 2. Technological scheme of the subsystem Fig. 3. Fault tree diagram and fault tree table (generated in Reliability Studio and extended manually in CorelDraw)

  table, fault tree table, subdiagrams/mirrored blocks/multiblocks, LCC, Weibull tree, throughtput analysis, downtime distributions, parts table, prediction data, FMEA worksheet, maintainability data, maintenance policies, RBD, fault tree diagram, event tree diagram, Weibull graph. (x.) Simultaneous analyses. This feature can be used to handle Availability, Reliability and Maintenance together. (xi.) Combining series and parallel subsystems.

Table 2

 2 Input values

	Measure	Quantity	2007	2008
	Time duration analysed (t)	h	8119.500	7058.000
	Total uptime (TUT)	h	7980.500	6909.718
	Total downtime (TDT)	h	139.000	148.282

Acknowledgements

The financial support from the EC project Marie Curie Chair (EXC) MEXC-CT-2003-042618 "Integrated Waste to Energy Management to Prevent Global Warming -INEMAGLOW" is gratefully acknowledged.

ACCEPTED MANUSCRIPT

The paper has been prepared in close collaboration and with an extensive support from MOL refinery. The authors would like to express a gratitude to all employees of the MOL Group, especially Dr. Gabriella Pécsvári, István Rabi and Zsolt Czaltig.