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Seasonality and circadian variation of microfilaremia in dogs experimentally infected with Dirofilaria immitis
Accepted Manuscript Title: Seasonality and circadian variation of microfilaremia in dogs experimentally infected with Dirofilaria immitis Authors: L´eonore Lovis, M´elanie Grandjean, Laurence Overney, Wolfgang Seewald, Heinz Sager PII: DOI: Reference:
S0304-4017(17)30309-6 http://dx.doi.org/doi:10.1016/j.vetpar.2017.07.010 VETPAR 8406
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
24-5-2017 3-7-2017 12-7-2017
Please cite this article as: Lovis, L´eonore, Grandjean, M´elanie, Overney, Laurence, Seewald, Wolfgang, Sager, Heinz, Seasonality and circadian variation of microfilaremia in dogs experimentally infected with Dirofilaria immitis.Veterinary Parasitology http://dx.doi.org/10.1016/j.vetpar.2017.07.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Seasonality and circadian variation of microfilaremia in dogs experimentally infected with Dirofilaria immitis
Léonore Lovis, Mélanie Grandjean, Laurence Overney, Wolfgang Seewald, Heinz Sager*
Elanco Animal Health, Switzerland
*Correspondence address: Heinz Sager, Elanco Animal Health, Schwarzwaldallee 215, CH-4058 Basel, Switzerland; Tel: +41 61 685 63 22; e-mail: [email protected]
1
Highlights
1
The changes in microfilaremia were observed for up to 3 years in 16 dogs experimentally infected with Dirofilaria immitis Seasonality was found in all dogs with microfilaremia-peaks in summer and 5 to 49-times lower counts in winter The circadian cycle of D. immitis microfilariae in the peripheral blood varied considerably between dogs and season
Abstract
Periodicity, the cyclical rise and fall in microfilaria (mff) numbers in the peripheral blood over time, is observed in many filarial infections. It is correlated with the necessity for these larval stages to be ingested by the blood feeding vector before they can be transmitted to a new vertebrate host. Microfilariae of the dog heartworm Dirofilaria immitis have been described to show periodicity, but the circadian pattern does not seem to be consistent. Most publications describe the lowest mff-concentrations in the peripheral blood in the early morning, while the highest counts occurred either in the afternoon, in the late evening or shortly after midnight. Sixteen dogs were experimentally infected with D. immitis isolates originating from Italy (one isolate, 14 dogs), and the USA (two isolates, one dog each). The dogs were housed indoors with a natural light source (windows) and heating that prevented temperature-drops below 20°C during winter. When patency was reached, blood samples were collected at weekly and monthly intervals over a period of up to 3 years, and at given hours of the day (morning, noon, evening) for the duration of one year in order to determine seasonal, as well as daily variations of microfilaremia.
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Despite the fact that the dogs were kept indoors, there was an apparent seasonality of the D. immitis-microfilaremia, with peaks in summer and 5 to 49-times lower counts in winter. This difference was statistically significant and the ratio remained constant over the years, regardless of the fact that the mff-counts increased from the first to the second year of patency. Since the temperature was kept constantly in a range between 20 to 26°C (with some single outliners in both directions) the climatic conditions may not explain this observation. Therefore, day length may be the most obvious reason for the seasonality in the given study set-up. Interestingly, the Italian D. immitis-isolate lost seasonality after three passages of experimental infections in dogs. The circadian cycle of mff in the peripheral blood varied considerably between dogs and season. There was no consistent or apparent pattern, which led to the conclusion that many individual factors seem to influence the appearance of mff in the peripheral blood, even, or especially, under standardized environmental conditions.
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Keywords
Dirofilaria immitis; dog; microfilaremia; seasonality; circadian periodicity
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Introduction
Microfilarial periodicity that comprises a cyclical rise and fall in microfilaria (mff) numbers over time in the peripheral blood is observed in many filarial infections (Sasa and Tanaka, 1972). This observation may be linked to the necessity of the parasites to be ingested by blood sucking arthropods in order to be 3
transmitted to a new vertebrate host (Hawking, 1967). Microfilariae of the dog heartworm Dirofilaria immitis exhibit a subperiodicity without clear nocturnal or diurnal peaks, where the wave pattern is present but mff do not completely disappear from the peripheral blood (Church et al., 1976). The physiologic basis for periodicity is unknown; however, the work of several investigators suggests that one or a variety of host factors such as oxygen-pressure in the lung or body temperature influence the periodic trend of some microfilaremias. (Hawking, 1967; Eberhard and Rabalais, 1976). Furthermore, it has been demonstrated for the northern hemisphere that D. immitis mff are most abundant in the peripheral blood during July and August, with increasing numbers starting in April and decreasing in October (Hawking, 1967; Newton, 1968). The periodicity seems to be linked to the presence of the respective vector mosquitoes of the family Culicidae (Ledesma and Harrington, 2011; Otranto et al., 2013). However, the mechanisms producing such seasonal variation are largely unknown. It was assumed that the longer daylight during summer affects some endocrine mechanisms in the dog which may stimulate the female filarial worms to produce greater numbers of microfilariae (Hawking, 1967). During our heartworm research programme, we had the opportunity to collect blood from experimentally infected D. immitis donor-dogs over a period of several years. This allowed observation of the changes in microfilaremia over several seasons in the same dog as well as the behavior of an isolate over time. In addition, it was possible to assess the circadian behavior over a period of one year.
4 4.1
Material and methods Animals and housing
4
Sixteen beagle dogs, aged 6 to 12 months, were experimentally infected with D. immitis in four separate studies. All dog experiments were performed in accordance with the Swiss Animal Protection Act and were approved by the cantonal veterinary authorities (permits FR 401/08E, 2010_46_FR and 2010_46_E4_FR). The dogs were group-housed in rooms with outdoor-access until the end of the prepatency period. Afterwards they were transferred to an indoor-facility with restricted access and mosquito-proof barriers. The temperature range was set to maintain values between 20 to 26°C, but exceeded these values temporarily during summer since there was a heating, without cooling, system installed. The rooms were illuminated by natural light which was supplemented by an artificial light-source from 06:00 am to 06:00 pm.
4.2
Infection and origin of the isolates
For experimental infection, three different D. immitis-isolates were used: (1) Ld2006, isolated 2006 in the Po river valley in Northern Italy, (2) Td2008, isolated 2008 in West Monroe, Louisiana, USA, and (3) Jd2009, isolated 2009 in West Memphis, Arkansas, USA. The Italian isolate was maintained at the Research Center of Novartis Animal Health, in St-Aubin, Switzerland since 2007 and both American isolates since 2009. The latter were further described by Bourguinat et al. (2015). For infection, approximately 50 third stage D. immitis larvae (L3) suspended in Hank's Balanced Salt Solution without phenol red were injected subcutaneously between the shoulder blades of the puppies. The L3 were obtained by feeding Aedes aegypti mosquitoes with microfilaremic blood of donor dogs. Table 1 summarizes the used isolates and infection dates for each dog.
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4.3
Blood collection
Blood was sampled from the jugular veins, using EDTA as an anticoagulant and was examined within one hour after collection. For assessment of the circadian cycle, 6 dogs (4 dogs infected with the Italian isolate Ld2006, 1 dog with Td2008 and 1 dog with Jd2009) were sampled 3 times a day at approximately 06:30 am, 12:00 pm and 06:30 pm over 5 consecutive days per month for the duration of one year (in total 180 blood samples were collected per dog). To address seasonal changes of microfilaremia of the Italian Ld-2006 isolate, blood samples were collected from 14 dogs at weekly or monthly intervals over a period of 1 to 3 years. Blood sampling was performed in the morning in order to be consistent and to minimize circadian fluctuation. Table 1 summarizes the sampling schedule for each dog. For counting of mff, 20 µL of blood were mixed with 100 µL of water and spread as a thin layer on a microscope slide. Microfilariae were then immediately counted with the help of a microscope. For each sampling time point, counts were performed on two aliquots and the arithmetic mean was calculated for each dog.
4.4
Statistical analysis
A three-way ANOVA was applied to evaluate seasonal changes based on monthly averages after log (base 10) transformation, with year, month and subject as model effects. All calculations were carried out using the software SAS®, Version 9.2.2 (Cary, NC, SAS Institute Inc.).
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5
Results
Figure 1 shows the mff counts of the four dogs from study N° 01 infected with the Italian D. immitis isolate Ld2006 and sampled weekly between November 2008 and January 2012. Seasonal variations in microfilaremia were observed each year in all dogs, with reduced counts in winter and peaks in summer. Microfilaremia started to increase in March/April, and declined in September/October. The ANOVA-analysis revealed significant differences between November through February on the one hand and May through September on the other hand (P < 0.001 for each pairwise comparison), with non-significant differences from November through February or from May through September. There was also a significant increase of microfilaremia from the first to the second year of patency (p <0.001). In the following year no significant changes in the overall microfilaremia were observed (p = 0.35). Seasonal variations were also observed for dogs from studies N° 02 and 03. In order to confirm the observation of increasing peaks between the first and the following patent years, the mff-counts of all dogs from studies N° 01, 02 and 03 infected with the Italian isolate Ld2006 were grouped (n = 8). The results obtained for the first year of patency (Figure 2a) and for the two subsequent years, i.e. year 2 and 3 (Figure 2b) confirmed the higher summer-counts for the latter. Despite low values in winter, mff were consistently detected in the blood and were higher in the second and third year compared to the first year of patency. As a consequence, the ratios between minimum (in November to February) and maximum mff-counts (in July/August) of each individual dog did not change considerably and stayed in a range of 12 to 49 in the first year and 5 to 39 in the following two years. In contrast, none of the six dogs of study N° 04 that were infected with L3 of the 3rd passage of Ld2006, showed the typical seasonal microfilaremia-pattern when assessed between April 2013 and November 2015 (Figure 3).
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No shared circadian pattern was observed for the 6 dogs of which blood samples were collected 3 times daily on 5 consecutive days per month between June 2010 and June 2011. The 4 dogs infected with the Italian D. immitis isolate Ld2006 did not show any consistent pattern at all (see Figure 4a, for dog 2657 as example). The two dogs infected with the American isolates demonstrated two different patterns, which changed through the seasons. For dog 2884, infected with Jd2009, the highest mff-counts were observed at 12:00 am from October to April, while they were highest at 06:30 am from May to September (Figure 4b). For dog 2886, infected with Td2008, the highest microfilaria counts were observed at 06:30 pm from June to December, while January was dominated by mff-peaks at 06:30 am, followed by a period without a consistent pattern (Figure 4c). The ratio between the minimal and maximal daily counts for the Italian isolate was in the range of 1 and 2.1, with some higher values for dog 2666 which ended up in a maximum-ratio of 3.8. The American isolates showed higher ratios of up to 6.1 and 6.7 for Jd2009 and Td2008, respectively.
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Discussion
Despite the fact that the patent dogs were housed indoors under defined climatic conditions, there was a clear tendency for seasonal variation of the number of mff found in the peripheral blood – at least for the first two passages of the respective D. immitis-isolates. This may be linked to day length, since the dogs were exposed to natural daylight. Hawking (1967) hypothesized a correlation between seasonality and day length. Under our experimental setup, an artificial light source was switched on between 06:00 and 18:00 to supplement the weaker luminosity during winter. Under this assumption it is possible that the reduction in microfilaremia between October and March may be even more pronounced, since the length of the day was artificially prolonged.
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The microfilaremia varied between the first and the following years of patency. It can be hypothesized that the adult females needed some time to reach full productivity, but it also seems that the microfilaremia reached a plateau in the second year. Furthermore, the ratio between minimum and maximum counts did not alter considerably over the years, i.e. the peaks in summer were higher, but also the mff-counts during winter were elevated in the second and third year compared to the first year of patency. The observation that after 3 passages the seasonal microfilaremia was lost, raises the question if the parasites adapted to the lab-conditions. Compared to field conditions, the dogs were less exposed to seasonal and daily temperature-changes. We observed in an earlier study a considerable decrease in microfilaremia during winter when the housing temperatures were below 20°C (data not shown). It remains unclear how the lower temperatures impact the microfilaremia, since the dogs maintain a constant body temperature. Nevertheless, it has been described by Eberhard and Rabalais (1976) that the body temperature may be a parameter influencing the number of mff in the peripheral blood. This was in the frame of circadian periodicity and linked with physical activity, but it could partially serve as an explanation for seasonal variations. Based on that experience, we intensified the heating during winter to maintain a median-temperature of 24°C with a range between 20 to 26°C with only a few excursions below 20°C for a few hours (minimum at 17.9°C). In summer, the temperatures were higher since no active cooling system was installed and may have exceeded 30°C on individual days. It remains unclear, if the relatively low difference between minimum and maximum temperature can be an explanation for loss of seasonality. Since the dogs were used for production of mff for experimental infection of mosquitos all year round, there may have been a selection of filariae that delivered high numbers of larvae during winter. From an evolutionary viewpoint it would make sense that the parasite can adapt to changes in vector behavior. But we are lacking proof that such a selection takes place within only two generations. Furthermore, it remains unclear if the seasonal periodicity is
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based on the fertility of the female worms or if the mff change their distribution-pattern in the peripheral blood. The former was proposed by Hawking (1967), but there is, to best of our knowledge, no further information in more recent literature. The observation of no consistent circadian pattern, at least for the Italian isolate Ld2006, is not in agreement with the literature. Many authors observed a circadian pattern, but there is a considerable variation throughout the different publications (Table 2). Rhee and colleagues (1998) mention geographical differences to explain the inconsistent circadian pattern. On the other hand side, despite all the differences, there is consistency in the way that the minimum mff-counts were mostly found between 04:00 am and 08:00 am, while the maximum-counts occurred between 04:00 pm and 08:00 pm (Table 2). The observation-period in most of these studies is rather short, i.e. one to a few days. Only Euzéby and Lainé (1951) who collected blood samples over one month. They observed considerable variation in the course of the month, but the pattern remained relatively consistent with minimum mff-counts at 08:00 am and maximum counts at 08:00 pm. The four dogs infected with the Ld2006-isolate also displayed a circadian pattern when looking only at defined time-windows of 2 to 3 days. But it would not have been consistent between the individuals, i.e. each dog had the maximum mff-counts at a different sampling point at a specific date. The dogs infected with the American isolates showed a consistent pattern over several months. Nevertheless, it remains unclear, if another dog infected with this isolate would have presented the same circadian pattern. In addition, the pattern between the Jd2009- and the Td2008-isolate differed considerably. Aoki described in his review-article a study performed by Yoshida, who examined the circadian pattern of mff in the peripheral blood of patients traveling from Okinawa to Bolivia on a passenger ship (Aoki et al., 2011). In that study, blood-samples were collected every week for 2 months. Interestingly, the periodicity of mff adapted to the light/dark cycle of the actual location (Aoki et al., 2011). Based on the data from our study, it is unlikely, that the circadian pattern of the two US-isolates was still impacted by their origin. Firstly, the entire first passage was done in Europe, i.e. the larval development in mosquitos, the infection of dogs with L3, the whole prepatency, and the patency-period occurred in the Central European-time zone.
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Secondly, as described by Aoki, the adaptation to a new time zone seems to happen very quickly. In our case, the sampling of blood started more than half a year, for Td2008 even more than a year after the isolation in the USA. In that regard there was sufficient time to adapt to the new light/dark-cycle (Aoki et al., 2011).
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Conclusion
The seasonality of D. immitis mff in the peripheral blood of dogs seems to be driven by the day length as one of the most important parameters. This was hypothesized in our experimental setup where the duration of light exposure was one of the few remaining variables to explain the seasonal change of microfilaremia. On the other hand, we are still lacking sufficient understanding of other control mechanisms, especially since the Italian D. immitis-isolate lost its seasonality after only three experimental passages. The circadian cycle of D. immitis microfilaremia cannot be accurately characterized as diurnal or nocturnal as shown in Table 2. By observing experimentally infected dogs over a period of one year, we can conclude for the used D. immitis-isolates that there is no consistent circadian pattern of microfilaremia and season does not seem to have an impact on the circadian peaks under the described experimental setup. Finally, it seems that there are many individual factors that influence the appearance of mff in the peripheral blood, even, or especially, under standardized environmental conditions.
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Acknowledgements
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The D. immitis isolates were obtained with the kind support of Prof. C. Genchi, University of Milan (Italy, Ld2006), and Prof. B. Blagburn, Auburn University, Alabama (USA, Td2008 and Jd2009). The authors would like to thank Dr. Regina Lizundia for critical review of the paper and Dr. R. Kaminsky and Dr. C. Epe for their contribution to the design of the studies.
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Conflict of interest
All experiments were performed at the Research site of Novartis Animal Health in St-Aubin (Switzerland), which is part of Elanco Animal Health. There was no funding from external parties.
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References Angus,;1; B.M.,;1; 1981. Periodicity exhibited by microfilariae of Dirofilaria immitis in South East Queensland. Australian Vet. J. 57, 101-102.
Aoki, Y., Fujimaki, Y., Isao Tada, I.,;1; 2011. Basic Studies on Filaria and Filariasis. Trop. Med. Health. 39 (Suppl 2), 51-55.
Bourguinat, C., Lee, A.C., Lizundia, R., Blagburn, B.L., Liotta, J.L., Kraus, M.S., Keller, K., Epe, C., Letourneau, L., Kleinman, C.L., Paterson, T., Gomez, E.C., Montoya-Alonso, J.A., Smith, H., Bhan, A., Peregrine, A.S., Carmichael, J., Drake, J., Schenker, R., Kaminsky, R., Bowman, D.D., Geary, T.G., Prichard, R.K.,;1; 2015. Macrocyclic lactone resistance in Dirofilaria immitis: Failure of heartworm preventives and investigation of genetic markers for resistance. Vet. Parasitol. 210, 167-178.
Eberhard, M.L., Rabalais, F.,;1; 1976. Dipetalonema viteae: effects of hypo- and hyperthermic stress on microfilaremia in the Mongolian jird, Meriones unguiculatus. Exp. Parasitol. 40, 5-12.
Euzéby, J., Lainé, B.,;1; 1951. Sur la périodicité des microfilaires de Dirofilaria immitis. Ses variations sous l’influence de divers facteurs. Revue Méd. Vét., 102, 231-238.
Grieve, R.B., Lauria, S.,;1; 1983. Periodicity of Dirofilaria immitis microfilariae in canine and murine hosts. Acta. Trop. 40, 121-127.
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Hawking, F.,;1; 1967. The 24-hour periodicity of microfilariae: biological mechanisms responsible for its production and control. Proc. Roy. Soc. B. 169, 5967.
Ionică, A.M., Matei, I.A., D'Amico, G., Bel, L.V., Dumitrache, M.O., Modrý, D., Mihalca, A.D.,;1; 2017. Dirofilaria immitis and D. repens show circadian co-periodicity in naturally co-infected dogs. Parasit. Vectors. 10, 116 (doi: 10.1186/s13071-017-2055-2).
Katamine, D.,;1; 1970. Studies on the periodicity of microfilariae. In: Sasa, M. (Ed), Recent Advances in Research on Filariasis and Schistosomiasis in Japan. Tokyo: University of Tokyo Press and University of Park Press, 123-144.
Ledesma, N., Harrington, L.,;1; 2011. Mosquito vectors of dog heartworm in the United States: vector status and factors influencing transmission efficiency. Top. Companion Anim. Med. 26, 178-185.
Matola, Y.G.,;1; 1991. Periodicity of Dirofilaria immitis microfilariae in a dog from Muheza district, Tanzania. J. Helminthology, 65, 76-78.
Newton, W.L.,;1; 1968. Longevity of an experimental infection with Dirofilaria immitis in a dog. J. Parasitol. 54, 187-188.
Otranto, D., Dantas-Torres, F., Brianti, E., Traversa, D., Petrić, D., Genchi, C., Capelli, G.,;1; 2013. Vector-borne helminths of dogs and humans in Europe. Parasit. Vectors. 6, 16 (doi: 10.1186/1756-3305-6-16).
Rhee, J.K., Yang, S.S., Kim, H.C.,;1; 1998. Periodicity exhibited by Dirofilaria immitis microfilariae identified in dogs of Korea. Korean J. Parasitol. 36, 235-239. 14
Sasa, M., Tanaka, H.,;1; 1972. Studies on the methods for statistical analysis of the microfilarial periodicity survey data. Southeast Asian J. Trop. Med. Publ. Hlth. 4, 518-536.
Schnelle, G.B., Young, R.M.,;1; 1944. Clinical studies on microfilarial periodicity in war dogs. Bull. US Army Med. Dept. 80, 52–59.
Underwood, P.C., Wright, H.W.,;1; 1933. Observations on the periodicity of Dirofilaria immitis larvae in the peripheral blood of dogs. Proceedings of the Helminthological Society of Washington the one hundred and fifty-fifth meeting. J. Parasitol. 20, 113.
Webber, W.A.F., Hawking, F.,;1; 1955. Experimental maintenance of Dirofilaria repens and D. immitis in dogs. Exp. Parasitol. 4, 143-164.
Figure captions Figure 1: Season-dependent microfilaremia of 4 dogs (identification number 2657, 2661, 2666 and 2670) infected with the Italian D. immitis-isolate Ld2006. The first blood sample was collected on 26-Nov-2008, the last on 10-Jan-2012.
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Figure 2: Microfilaremia of 8 dogs experimentally infected with the D. immitis-isolate Ld2006, in the first year of parasitaemia (year 1; A) and in the following two years (year 2 and 3 were combined; B). The box-plots represent the 1st, 2nd and 3rd quartile, the vertical bars mark the minimum and maximum values. The expected day length is depicted in C. The horizontal line indicates the duration of artificial light exposure, i.e. between 06:00 am and 06:00 pm.
Figure 3: Microfilaremia of 6 dogs experimentally infected with the D. immitis-isolate Ld2006 at the third passage. Blood samples were collected on a monthly basis. The box-plots represent the 1st, 2nd and 3rd quartile, the vertical bars mark the minimum and maximum values of microfilaremia.
Figure 4: Circadian periodicity of microfilaremia over the period of one year. Three blood samples per day were collected from each dog, early in the morning (~06:30 am), at noon (~12:00 am) and in the evening (~06:30 pm). The microfilaremia in the morning (dashed line) and the evening (dotted line) is expressed as ratio in comparison to the mff-counts at noon (horizontal grey line). Three examples of individual dogs are shown: Dog 2657, experimentally infected with D. immitis isolate Ld2006 (A), dog 2884, infected with the isolate Jd2009 (B) and dog 2886, infected with isolate Td2008 (C).
Tables
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Table 1: Information on D. immitis-isolate, infection, dates and frequency of blood sampling from each dog.
Dog
Infection
Blood sampling
Study N° N°
Sex
2657
M*
2661
M*
2666
F
Isolate
Passage
Date
Ld2006
2
08 Apr 2008
Ld2006
1
08 Apr 2008
Dates
Frequency
Nov 2008 - Jan 2012 /
Weekly
(Jun 2010 - Jun 2011)
(3x daily)
Nov 2008 - Jan 2012 /
Weekly
(Jun 2010 - Jun 2011)
(3x daily)
/
01
02
2670
F
2822
F
Ld2006
2
04 Sep 2008
Apr 2009 - Aug 2010
weekly
2884
F
Jd2009
1
16 Nov 2009
Jun 2010 - Jun 2011
3x daily
2886
F
Td2008
1
17 Sep 2009
Jun 2010 - Jun 2011
3x daily
2923
M*
Ld2006
2
26 Jan 2010
Apr 2013 - Jan 2015
monthly
2924
F Ld2006
2
26 Jan 2010
Aug 2010 - Sep 2011 /
Weekly
Apr 2013 - Jan2015
monthly
/
03
2925
M*
17
/
11098
F
11099
M*
11100
F
11101
M*
11102
F
11103
M*
04
Ld2006
3
Apr 2013 - Nov 2015
monthly
Apr 2013 - Jul 2015
monthly
Apr 2013 - Nov 2015
monthly
14 Mar 2012
* Male dogs were castrated before entry into the study.
Table 2: Overview of scientific publications on circadian periodicity of microfilaremia in D. immitis infected dogs
Country
Sampling period
interval
Number
Type of
Sampling time (hour of the day; intervals cover 2 hours-periods)
of dogs
infection
02
04
06
08
10
12
02
04
06
08
10
12
am
am
am
am
am
am
pm
pm
pm
pm
pm
pm
mi
mi
MA
MA
Korea
72h
2h
10
natural
Australia
24h
1-2h
3
natural
Tanzania
5 days
1h
1
natural
MA
mi
mi
MA MA
18
mi
Ratio
Reference
2
Rhee et al., 1998
mi
3-7
Angus, 1981
mi
3-4
Matola, 1991
USA
48h
4h
1
Exp.
NA
30h
2-4h
NA
NA
NA
24h
2h
2
NA
MA
MA
MA
mi
MA
MA
mi
MA
3-4
Grieve and Lauria, 1983
2-5
Hawking, 1967
NA
Underwood
and
Wright,
1932 China1
28h
(2)-4h
1
Exp.
France
1 month
8h, 12h,
1
NA
mi
mi
5-20
Webber and Hawking, 1955
3-8
Euzéby and Lainé, 1951
MA
NA
Katamine, 1970
mi
NA
Schnelle and Young, 1944
NA
Ionică et al., 2017
MA mi
MA
20h, 24h Japan
24h
2h
3
NA
USA
14h
2h
4
natural
Romania
48 h
2h
4
natural2
1
mi mi MA
mi
MA MA
mi
The isolate originally came from China, but the experimental infection of the dogs and the blood sampling was done in UK; 2 Co-infection of D. immitis and
D. repens. Abbreviations: Ratio: ratio between maximum and minimum microfilariae-counts; mi: minimum microfilaremia; MA: maximum microfilaremia; Exp.: experimental infection; NA: not applicable, no information available.
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S0304-4017(17)30309-6 http://dx.doi.org/doi:10.1016/j.vetpar.2017.07.010 VETPAR 8406
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
24-5-2017 3-7-2017 12-7-2017
Please cite this article as: Lovis, L´eonore, Grandjean, M´elanie, Overney, Laurence, Seewald, Wolfgang, Sager, Heinz, Seasonality and circadian variation of microfilaremia in dogs experimentally infected with Dirofilaria immitis.Veterinary Parasitology http://dx.doi.org/10.1016/j.vetpar.2017.07.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Seasonality and circadian variation of microfilaremia in dogs experimentally infected with Dirofilaria immitis
Léonore Lovis, Mélanie Grandjean, Laurence Overney, Wolfgang Seewald, Heinz Sager*
Elanco Animal Health, Switzerland
*Correspondence address: Heinz Sager, Elanco Animal Health, Schwarzwaldallee 215, CH-4058 Basel, Switzerland; Tel: +41 61 685 63 22; e-mail: [email protected]
1
Highlights
1
The changes in microfilaremia were observed for up to 3 years in 16 dogs experimentally infected with Dirofilaria immitis Seasonality was found in all dogs with microfilaremia-peaks in summer and 5 to 49-times lower counts in winter The circadian cycle of D. immitis microfilariae in the peripheral blood varied considerably between dogs and season
Abstract
Periodicity, the cyclical rise and fall in microfilaria (mff) numbers in the peripheral blood over time, is observed in many filarial infections. It is correlated with the necessity for these larval stages to be ingested by the blood feeding vector before they can be transmitted to a new vertebrate host. Microfilariae of the dog heartworm Dirofilaria immitis have been described to show periodicity, but the circadian pattern does not seem to be consistent. Most publications describe the lowest mff-concentrations in the peripheral blood in the early morning, while the highest counts occurred either in the afternoon, in the late evening or shortly after midnight. Sixteen dogs were experimentally infected with D. immitis isolates originating from Italy (one isolate, 14 dogs), and the USA (two isolates, one dog each). The dogs were housed indoors with a natural light source (windows) and heating that prevented temperature-drops below 20°C during winter. When patency was reached, blood samples were collected at weekly and monthly intervals over a period of up to 3 years, and at given hours of the day (morning, noon, evening) for the duration of one year in order to determine seasonal, as well as daily variations of microfilaremia.
2
Despite the fact that the dogs were kept indoors, there was an apparent seasonality of the D. immitis-microfilaremia, with peaks in summer and 5 to 49-times lower counts in winter. This difference was statistically significant and the ratio remained constant over the years, regardless of the fact that the mff-counts increased from the first to the second year of patency. Since the temperature was kept constantly in a range between 20 to 26°C (with some single outliners in both directions) the climatic conditions may not explain this observation. Therefore, day length may be the most obvious reason for the seasonality in the given study set-up. Interestingly, the Italian D. immitis-isolate lost seasonality after three passages of experimental infections in dogs. The circadian cycle of mff in the peripheral blood varied considerably between dogs and season. There was no consistent or apparent pattern, which led to the conclusion that many individual factors seem to influence the appearance of mff in the peripheral blood, even, or especially, under standardized environmental conditions.
2
Keywords
Dirofilaria immitis; dog; microfilaremia; seasonality; circadian periodicity
3
Introduction
Microfilarial periodicity that comprises a cyclical rise and fall in microfilaria (mff) numbers over time in the peripheral blood is observed in many filarial infections (Sasa and Tanaka, 1972). This observation may be linked to the necessity of the parasites to be ingested by blood sucking arthropods in order to be 3
transmitted to a new vertebrate host (Hawking, 1967). Microfilariae of the dog heartworm Dirofilaria immitis exhibit a subperiodicity without clear nocturnal or diurnal peaks, where the wave pattern is present but mff do not completely disappear from the peripheral blood (Church et al., 1976). The physiologic basis for periodicity is unknown; however, the work of several investigators suggests that one or a variety of host factors such as oxygen-pressure in the lung or body temperature influence the periodic trend of some microfilaremias. (Hawking, 1967; Eberhard and Rabalais, 1976). Furthermore, it has been demonstrated for the northern hemisphere that D. immitis mff are most abundant in the peripheral blood during July and August, with increasing numbers starting in April and decreasing in October (Hawking, 1967; Newton, 1968). The periodicity seems to be linked to the presence of the respective vector mosquitoes of the family Culicidae (Ledesma and Harrington, 2011; Otranto et al., 2013). However, the mechanisms producing such seasonal variation are largely unknown. It was assumed that the longer daylight during summer affects some endocrine mechanisms in the dog which may stimulate the female filarial worms to produce greater numbers of microfilariae (Hawking, 1967). During our heartworm research programme, we had the opportunity to collect blood from experimentally infected D. immitis donor-dogs over a period of several years. This allowed observation of the changes in microfilaremia over several seasons in the same dog as well as the behavior of an isolate over time. In addition, it was possible to assess the circadian behavior over a period of one year.
4 4.1
Material and methods Animals and housing
4
Sixteen beagle dogs, aged 6 to 12 months, were experimentally infected with D. immitis in four separate studies. All dog experiments were performed in accordance with the Swiss Animal Protection Act and were approved by the cantonal veterinary authorities (permits FR 401/08E, 2010_46_FR and 2010_46_E4_FR). The dogs were group-housed in rooms with outdoor-access until the end of the prepatency period. Afterwards they were transferred to an indoor-facility with restricted access and mosquito-proof barriers. The temperature range was set to maintain values between 20 to 26°C, but exceeded these values temporarily during summer since there was a heating, without cooling, system installed. The rooms were illuminated by natural light which was supplemented by an artificial light-source from 06:00 am to 06:00 pm.
4.2
Infection and origin of the isolates
For experimental infection, three different D. immitis-isolates were used: (1) Ld2006, isolated 2006 in the Po river valley in Northern Italy, (2) Td2008, isolated 2008 in West Monroe, Louisiana, USA, and (3) Jd2009, isolated 2009 in West Memphis, Arkansas, USA. The Italian isolate was maintained at the Research Center of Novartis Animal Health, in St-Aubin, Switzerland since 2007 and both American isolates since 2009. The latter were further described by Bourguinat et al. (2015). For infection, approximately 50 third stage D. immitis larvae (L3) suspended in Hank's Balanced Salt Solution without phenol red were injected subcutaneously between the shoulder blades of the puppies. The L3 were obtained by feeding Aedes aegypti mosquitoes with microfilaremic blood of donor dogs. Table 1 summarizes the used isolates and infection dates for each dog.
5
4.3
Blood collection
Blood was sampled from the jugular veins, using EDTA as an anticoagulant and was examined within one hour after collection. For assessment of the circadian cycle, 6 dogs (4 dogs infected with the Italian isolate Ld2006, 1 dog with Td2008 and 1 dog with Jd2009) were sampled 3 times a day at approximately 06:30 am, 12:00 pm and 06:30 pm over 5 consecutive days per month for the duration of one year (in total 180 blood samples were collected per dog). To address seasonal changes of microfilaremia of the Italian Ld-2006 isolate, blood samples were collected from 14 dogs at weekly or monthly intervals over a period of 1 to 3 years. Blood sampling was performed in the morning in order to be consistent and to minimize circadian fluctuation. Table 1 summarizes the sampling schedule for each dog. For counting of mff, 20 µL of blood were mixed with 100 µL of water and spread as a thin layer on a microscope slide. Microfilariae were then immediately counted with the help of a microscope. For each sampling time point, counts were performed on two aliquots and the arithmetic mean was calculated for each dog.
4.4
Statistical analysis
A three-way ANOVA was applied to evaluate seasonal changes based on monthly averages after log (base 10) transformation, with year, month and subject as model effects. All calculations were carried out using the software SAS®, Version 9.2.2 (Cary, NC, SAS Institute Inc.).
6
5
Results
Figure 1 shows the mff counts of the four dogs from study N° 01 infected with the Italian D. immitis isolate Ld2006 and sampled weekly between November 2008 and January 2012. Seasonal variations in microfilaremia were observed each year in all dogs, with reduced counts in winter and peaks in summer. Microfilaremia started to increase in March/April, and declined in September/October. The ANOVA-analysis revealed significant differences between November through February on the one hand and May through September on the other hand (P < 0.001 for each pairwise comparison), with non-significant differences from November through February or from May through September. There was also a significant increase of microfilaremia from the first to the second year of patency (p <0.001). In the following year no significant changes in the overall microfilaremia were observed (p = 0.35). Seasonal variations were also observed for dogs from studies N° 02 and 03. In order to confirm the observation of increasing peaks between the first and the following patent years, the mff-counts of all dogs from studies N° 01, 02 and 03 infected with the Italian isolate Ld2006 were grouped (n = 8). The results obtained for the first year of patency (Figure 2a) and for the two subsequent years, i.e. year 2 and 3 (Figure 2b) confirmed the higher summer-counts for the latter. Despite low values in winter, mff were consistently detected in the blood and were higher in the second and third year compared to the first year of patency. As a consequence, the ratios between minimum (in November to February) and maximum mff-counts (in July/August) of each individual dog did not change considerably and stayed in a range of 12 to 49 in the first year and 5 to 39 in the following two years. In contrast, none of the six dogs of study N° 04 that were infected with L3 of the 3rd passage of Ld2006, showed the typical seasonal microfilaremia-pattern when assessed between April 2013 and November 2015 (Figure 3).
7
No shared circadian pattern was observed for the 6 dogs of which blood samples were collected 3 times daily on 5 consecutive days per month between June 2010 and June 2011. The 4 dogs infected with the Italian D. immitis isolate Ld2006 did not show any consistent pattern at all (see Figure 4a, for dog 2657 as example). The two dogs infected with the American isolates demonstrated two different patterns, which changed through the seasons. For dog 2884, infected with Jd2009, the highest mff-counts were observed at 12:00 am from October to April, while they were highest at 06:30 am from May to September (Figure 4b). For dog 2886, infected with Td2008, the highest microfilaria counts were observed at 06:30 pm from June to December, while January was dominated by mff-peaks at 06:30 am, followed by a period without a consistent pattern (Figure 4c). The ratio between the minimal and maximal daily counts for the Italian isolate was in the range of 1 and 2.1, with some higher values for dog 2666 which ended up in a maximum-ratio of 3.8. The American isolates showed higher ratios of up to 6.1 and 6.7 for Jd2009 and Td2008, respectively.
6
Discussion
Despite the fact that the patent dogs were housed indoors under defined climatic conditions, there was a clear tendency for seasonal variation of the number of mff found in the peripheral blood – at least for the first two passages of the respective D. immitis-isolates. This may be linked to day length, since the dogs were exposed to natural daylight. Hawking (1967) hypothesized a correlation between seasonality and day length. Under our experimental setup, an artificial light source was switched on between 06:00 and 18:00 to supplement the weaker luminosity during winter. Under this assumption it is possible that the reduction in microfilaremia between October and March may be even more pronounced, since the length of the day was artificially prolonged.
8
The microfilaremia varied between the first and the following years of patency. It can be hypothesized that the adult females needed some time to reach full productivity, but it also seems that the microfilaremia reached a plateau in the second year. Furthermore, the ratio between minimum and maximum counts did not alter considerably over the years, i.e. the peaks in summer were higher, but also the mff-counts during winter were elevated in the second and third year compared to the first year of patency. The observation that after 3 passages the seasonal microfilaremia was lost, raises the question if the parasites adapted to the lab-conditions. Compared to field conditions, the dogs were less exposed to seasonal and daily temperature-changes. We observed in an earlier study a considerable decrease in microfilaremia during winter when the housing temperatures were below 20°C (data not shown). It remains unclear how the lower temperatures impact the microfilaremia, since the dogs maintain a constant body temperature. Nevertheless, it has been described by Eberhard and Rabalais (1976) that the body temperature may be a parameter influencing the number of mff in the peripheral blood. This was in the frame of circadian periodicity and linked with physical activity, but it could partially serve as an explanation for seasonal variations. Based on that experience, we intensified the heating during winter to maintain a median-temperature of 24°C with a range between 20 to 26°C with only a few excursions below 20°C for a few hours (minimum at 17.9°C). In summer, the temperatures were higher since no active cooling system was installed and may have exceeded 30°C on individual days. It remains unclear, if the relatively low difference between minimum and maximum temperature can be an explanation for loss of seasonality. Since the dogs were used for production of mff for experimental infection of mosquitos all year round, there may have been a selection of filariae that delivered high numbers of larvae during winter. From an evolutionary viewpoint it would make sense that the parasite can adapt to changes in vector behavior. But we are lacking proof that such a selection takes place within only two generations. Furthermore, it remains unclear if the seasonal periodicity is
9
based on the fertility of the female worms or if the mff change their distribution-pattern in the peripheral blood. The former was proposed by Hawking (1967), but there is, to best of our knowledge, no further information in more recent literature. The observation of no consistent circadian pattern, at least for the Italian isolate Ld2006, is not in agreement with the literature. Many authors observed a circadian pattern, but there is a considerable variation throughout the different publications (Table 2). Rhee and colleagues (1998) mention geographical differences to explain the inconsistent circadian pattern. On the other hand side, despite all the differences, there is consistency in the way that the minimum mff-counts were mostly found between 04:00 am and 08:00 am, while the maximum-counts occurred between 04:00 pm and 08:00 pm (Table 2). The observation-period in most of these studies is rather short, i.e. one to a few days. Only Euzéby and Lainé (1951) who collected blood samples over one month. They observed considerable variation in the course of the month, but the pattern remained relatively consistent with minimum mff-counts at 08:00 am and maximum counts at 08:00 pm. The four dogs infected with the Ld2006-isolate also displayed a circadian pattern when looking only at defined time-windows of 2 to 3 days. But it would not have been consistent between the individuals, i.e. each dog had the maximum mff-counts at a different sampling point at a specific date. The dogs infected with the American isolates showed a consistent pattern over several months. Nevertheless, it remains unclear, if another dog infected with this isolate would have presented the same circadian pattern. In addition, the pattern between the Jd2009- and the Td2008-isolate differed considerably. Aoki described in his review-article a study performed by Yoshida, who examined the circadian pattern of mff in the peripheral blood of patients traveling from Okinawa to Bolivia on a passenger ship (Aoki et al., 2011). In that study, blood-samples were collected every week for 2 months. Interestingly, the periodicity of mff adapted to the light/dark cycle of the actual location (Aoki et al., 2011). Based on the data from our study, it is unlikely, that the circadian pattern of the two US-isolates was still impacted by their origin. Firstly, the entire first passage was done in Europe, i.e. the larval development in mosquitos, the infection of dogs with L3, the whole prepatency, and the patency-period occurred in the Central European-time zone.
10
Secondly, as described by Aoki, the adaptation to a new time zone seems to happen very quickly. In our case, the sampling of blood started more than half a year, for Td2008 even more than a year after the isolation in the USA. In that regard there was sufficient time to adapt to the new light/dark-cycle (Aoki et al., 2011).
7
Conclusion
The seasonality of D. immitis mff in the peripheral blood of dogs seems to be driven by the day length as one of the most important parameters. This was hypothesized in our experimental setup where the duration of light exposure was one of the few remaining variables to explain the seasonal change of microfilaremia. On the other hand, we are still lacking sufficient understanding of other control mechanisms, especially since the Italian D. immitis-isolate lost its seasonality after only three experimental passages. The circadian cycle of D. immitis microfilaremia cannot be accurately characterized as diurnal or nocturnal as shown in Table 2. By observing experimentally infected dogs over a period of one year, we can conclude for the used D. immitis-isolates that there is no consistent circadian pattern of microfilaremia and season does not seem to have an impact on the circadian peaks under the described experimental setup. Finally, it seems that there are many individual factors that influence the appearance of mff in the peripheral blood, even, or especially, under standardized environmental conditions.
8
Acknowledgements
11
The D. immitis isolates were obtained with the kind support of Prof. C. Genchi, University of Milan (Italy, Ld2006), and Prof. B. Blagburn, Auburn University, Alabama (USA, Td2008 and Jd2009). The authors would like to thank Dr. Regina Lizundia for critical review of the paper and Dr. R. Kaminsky and Dr. C. Epe for their contribution to the design of the studies.
9
Conflict of interest
All experiments were performed at the Research site of Novartis Animal Health in St-Aubin (Switzerland), which is part of Elanco Animal Health. There was no funding from external parties.
12
References Angus,;1; B.M.,;1; 1981. Periodicity exhibited by microfilariae of Dirofilaria immitis in South East Queensland. Australian Vet. J. 57, 101-102.
Aoki, Y., Fujimaki, Y., Isao Tada, I.,;1; 2011. Basic Studies on Filaria and Filariasis. Trop. Med. Health. 39 (Suppl 2), 51-55.
Bourguinat, C., Lee, A.C., Lizundia, R., Blagburn, B.L., Liotta, J.L., Kraus, M.S., Keller, K., Epe, C., Letourneau, L., Kleinman, C.L., Paterson, T., Gomez, E.C., Montoya-Alonso, J.A., Smith, H., Bhan, A., Peregrine, A.S., Carmichael, J., Drake, J., Schenker, R., Kaminsky, R., Bowman, D.D., Geary, T.G., Prichard, R.K.,;1; 2015. Macrocyclic lactone resistance in Dirofilaria immitis: Failure of heartworm preventives and investigation of genetic markers for resistance. Vet. Parasitol. 210, 167-178.
Eberhard, M.L., Rabalais, F.,;1; 1976. Dipetalonema viteae: effects of hypo- and hyperthermic stress on microfilaremia in the Mongolian jird, Meriones unguiculatus. Exp. Parasitol. 40, 5-12.
Euzéby, J., Lainé, B.,;1; 1951. Sur la périodicité des microfilaires de Dirofilaria immitis. Ses variations sous l’influence de divers facteurs. Revue Méd. Vét., 102, 231-238.
Grieve, R.B., Lauria, S.,;1; 1983. Periodicity of Dirofilaria immitis microfilariae in canine and murine hosts. Acta. Trop. 40, 121-127.
13
Hawking, F.,;1; 1967. The 24-hour periodicity of microfilariae: biological mechanisms responsible for its production and control. Proc. Roy. Soc. B. 169, 5967.
Ionică, A.M., Matei, I.A., D'Amico, G., Bel, L.V., Dumitrache, M.O., Modrý, D., Mihalca, A.D.,;1; 2017. Dirofilaria immitis and D. repens show circadian co-periodicity in naturally co-infected dogs. Parasit. Vectors. 10, 116 (doi: 10.1186/s13071-017-2055-2).
Katamine, D.,;1; 1970. Studies on the periodicity of microfilariae. In: Sasa, M. (Ed), Recent Advances in Research on Filariasis and Schistosomiasis in Japan. Tokyo: University of Tokyo Press and University of Park Press, 123-144.
Ledesma, N., Harrington, L.,;1; 2011. Mosquito vectors of dog heartworm in the United States: vector status and factors influencing transmission efficiency. Top. Companion Anim. Med. 26, 178-185.
Matola, Y.G.,;1; 1991. Periodicity of Dirofilaria immitis microfilariae in a dog from Muheza district, Tanzania. J. Helminthology, 65, 76-78.
Newton, W.L.,;1; 1968. Longevity of an experimental infection with Dirofilaria immitis in a dog. J. Parasitol. 54, 187-188.
Otranto, D., Dantas-Torres, F., Brianti, E., Traversa, D., Petrić, D., Genchi, C., Capelli, G.,;1; 2013. Vector-borne helminths of dogs and humans in Europe. Parasit. Vectors. 6, 16 (doi: 10.1186/1756-3305-6-16).
Rhee, J.K., Yang, S.S., Kim, H.C.,;1; 1998. Periodicity exhibited by Dirofilaria immitis microfilariae identified in dogs of Korea. Korean J. Parasitol. 36, 235-239. 14
Sasa, M., Tanaka, H.,;1; 1972. Studies on the methods for statistical analysis of the microfilarial periodicity survey data. Southeast Asian J. Trop. Med. Publ. Hlth. 4, 518-536.
Schnelle, G.B., Young, R.M.,;1; 1944. Clinical studies on microfilarial periodicity in war dogs. Bull. US Army Med. Dept. 80, 52–59.
Underwood, P.C., Wright, H.W.,;1; 1933. Observations on the periodicity of Dirofilaria immitis larvae in the peripheral blood of dogs. Proceedings of the Helminthological Society of Washington the one hundred and fifty-fifth meeting. J. Parasitol. 20, 113.
Webber, W.A.F., Hawking, F.,;1; 1955. Experimental maintenance of Dirofilaria repens and D. immitis in dogs. Exp. Parasitol. 4, 143-164.
Figure captions Figure 1: Season-dependent microfilaremia of 4 dogs (identification number 2657, 2661, 2666 and 2670) infected with the Italian D. immitis-isolate Ld2006. The first blood sample was collected on 26-Nov-2008, the last on 10-Jan-2012.
15
Figure 2: Microfilaremia of 8 dogs experimentally infected with the D. immitis-isolate Ld2006, in the first year of parasitaemia (year 1; A) and in the following two years (year 2 and 3 were combined; B). The box-plots represent the 1st, 2nd and 3rd quartile, the vertical bars mark the minimum and maximum values. The expected day length is depicted in C. The horizontal line indicates the duration of artificial light exposure, i.e. between 06:00 am and 06:00 pm.
Figure 3: Microfilaremia of 6 dogs experimentally infected with the D. immitis-isolate Ld2006 at the third passage. Blood samples were collected on a monthly basis. The box-plots represent the 1st, 2nd and 3rd quartile, the vertical bars mark the minimum and maximum values of microfilaremia.
Figure 4: Circadian periodicity of microfilaremia over the period of one year. Three blood samples per day were collected from each dog, early in the morning (~06:30 am), at noon (~12:00 am) and in the evening (~06:30 pm). The microfilaremia in the morning (dashed line) and the evening (dotted line) is expressed as ratio in comparison to the mff-counts at noon (horizontal grey line). Three examples of individual dogs are shown: Dog 2657, experimentally infected with D. immitis isolate Ld2006 (A), dog 2884, infected with the isolate Jd2009 (B) and dog 2886, infected with isolate Td2008 (C).
Tables
16
Table 1: Information on D. immitis-isolate, infection, dates and frequency of blood sampling from each dog.
Dog
Infection
Blood sampling
Study N° N°
Sex
2657
M*
2661
M*
2666
F
Isolate
Passage
Date
Ld2006
2
08 Apr 2008
Ld2006
1
08 Apr 2008
Dates
Frequency
Nov 2008 - Jan 2012 /
Weekly
(Jun 2010 - Jun 2011)
(3x daily)
Nov 2008 - Jan 2012 /
Weekly
(Jun 2010 - Jun 2011)
(3x daily)
/
01
02
2670
F
2822
F
Ld2006
2
04 Sep 2008
Apr 2009 - Aug 2010
weekly
2884
F
Jd2009
1
16 Nov 2009
Jun 2010 - Jun 2011
3x daily
2886
F
Td2008
1
17 Sep 2009
Jun 2010 - Jun 2011
3x daily
2923
M*
Ld2006
2
26 Jan 2010
Apr 2013 - Jan 2015
monthly
2924
F Ld2006
2
26 Jan 2010
Aug 2010 - Sep 2011 /
Weekly
Apr 2013 - Jan2015
monthly
/
03
2925
M*
17
/
11098
F
11099
M*
11100
F
11101
M*
11102
F
11103
M*
04
Ld2006
3
Apr 2013 - Nov 2015
monthly
Apr 2013 - Jul 2015
monthly
Apr 2013 - Nov 2015
monthly
14 Mar 2012
* Male dogs were castrated before entry into the study.
Table 2: Overview of scientific publications on circadian periodicity of microfilaremia in D. immitis infected dogs
Country
Sampling period
interval
Number
Type of
Sampling time (hour of the day; intervals cover 2 hours-periods)
of dogs
infection
02
04
06
08
10
12
02
04
06
08
10
12
am
am
am
am
am
am
pm
pm
pm
pm
pm
pm
mi
mi
MA
MA
Korea
72h
2h
10
natural
Australia
24h
1-2h
3
natural
Tanzania
5 days
1h
1
natural
MA
mi
mi
MA MA
18
mi
Ratio
Reference
2
Rhee et al., 1998
mi
3-7
Angus, 1981
mi
3-4
Matola, 1991
USA
48h
4h
1
Exp.
NA
30h
2-4h
NA
NA
NA
24h
2h
2
NA
MA
MA
MA
mi
MA
MA
mi
MA
3-4
Grieve and Lauria, 1983
2-5
Hawking, 1967
NA
Underwood
and
Wright,
1932 China1
28h
(2)-4h
1
Exp.
France
1 month
8h, 12h,
1
NA
mi
mi
5-20
Webber and Hawking, 1955
3-8
Euzéby and Lainé, 1951
MA
NA
Katamine, 1970
mi
NA
Schnelle and Young, 1944
NA
Ionică et al., 2017
MA mi
MA
20h, 24h Japan
24h
2h
3
NA
USA
14h
2h
4
natural
Romania
48 h
2h
4
natural2
1
mi mi MA
mi
MA MA
mi
The isolate originally came from China, but the experimental infection of the dogs and the blood sampling was done in UK; 2 Co-infection of D. immitis and
D. repens. Abbreviations: Ratio: ratio between maximum and minimum microfilariae-counts; mi: minimum microfilaremia; MA: maximum microfilaremia; Exp.: experimental infection; NA: not applicable, no information available.
19