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Keywords:

  • modified live vaccines;
  • Bluetongue virus serotype 2;
  • Bluetongue virus serotype 9;
  • abortion;
  • pregnancy;
  • malformation;
  • livestock

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion and Conclusion
  7. Acknowledgements
  8. References

The recent outbreak caused by Schmallenberg virus, which affected sheep, goats and cattle in Europe, highlighted the importance of having a robust surveillance plan capable of monitoring abortions and malformations in the livestock offspring. In this context, bluetongue viruses (BTVs) represented and represent one of the major threats to the European livestock industry. Aiming to improve the understanding on BTV cross placental transmission and serotype involvement, in this retrospective study foetal spleens and/or brains of 663 ovines, 429 bovines, 155 goats and 17 buffaloes were tested for the presence of BTV by virus isolation. BTV vaccine strains were isolated from 31 foetuses (2.4%; 95% CI: 1.7–3.4%): 24 (3.6%; 95% CI: 2.4–5.3%) from ovine foetal tissues; 6 (1.4%; 95% CI: 0.6–3.0%) from bovine foetal tissues and 1 (0.6%; 95% CI: 0.2–3.5%) from the spleen of a caprine foetus. All foetuses were from animals vaccinated with either BTV-2 or BTV-2, and BTV-9 modified live vaccines (MLVs) produced by Onderstepoort Biological Products (OBP), South Africa. Among the 31 isolated vaccine strains, serotype 9 (n = 28) was more frequently isolated (P < 0.05) than serotype 2 (n = 3). In two cases infectious vaccine strains were found in the foetal tissues 2 months after the vaccine administration. Other pathogens known to be causative agents of abortion in ruminants were not detected nor isolated. This study demonstrates, for the first time, that BTV-2 and BTV-9 vaccine strains are able to cross the placental barrier of sheep, cattle and goats. BTV-2 and BTV-9 vaccine strains are able to infect foetuses and cause abortions or malformations depending on the period of pregnancy at the time of vaccination.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion and Conclusion
  7. Acknowledgements
  8. References

Bluetongue (BT) is an infectious vector-borne disease of ruminants caused by bluetongue virus (BTV), an RNA-virus which belongs to the family Reoviridae, genus Orbivirus (Verwoerd and Erasmus, 2004). At present, BTV officially includes 24 distinct serotypes (Verwoerd and Erasmus, 2004) based upon the immunological properties of the VP2 protein. The putative 25th and 26th serotypes have been proposed by Maan et al. (2011) and Chaignat et al. (2008). The disease is evident in sheep although clinical cases were reported in cattle (Toussaint et al., 2006).

The recent European BTV-8 outbreaks demonstrated for the first time the capability of the BTV-8 field strain to cross the placental barrier, causing the birth of viraemic offspring (De Clercq et al., 2008). Before the BTV-8 outbreak, vertical transmission had been observed only for BTV vaccine or tissue culture-adapted strains. All the reported BTV foetal infections had been associated to BTV-1, BTV-10, BTV-11 and BTV-17 vaccine strains (Johnson et al., 1992; Melville and Gard, 1992; MacLachlan et al., 2000; Kirkland and Hawkes, 2004). Since 2000, Italy has been experiencing the most severe outbreaks of BT ever recorded. The Italian government starting from May 2002 implemented a compulsory BTV vaccination campaign involving all susceptible domestic ruminants, in an attempt to reduce direct losses because of disease and indirect losses because of virus circulation. Prior to 2005, only modified live vaccines (MLV) produced by Onderstepoort Biological Products (OBP) in South Africa were employed. The MLV used were based on the serotypes present at that time in a given area. The aim of this study was to investigate whether BTV-2 and BTV-9 vaccine strains are capable of crossing the placental barrier of sheep, cattle, goat and buffaloes and to describe their effects on foetuses. This is the first report that demonstrates a correlation between BTV-2/9 MLV and reproductive failures in domestic ruminants under field conditions.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion and Conclusion
  7. Acknowledgements
  8. References

The study was conducted on foetuses aborted by animals following the 2003 Italian BT vaccination campaign, which used the combination of the monovalent BTV-2 and BTV-9 MLV produced by OBP. A total of 1272 foetuses and malformed offspring from sheep (n = 669), cattle (n = 431), goats (n = 155) and buffaloes (n = 17) were tested and processed for the most common abortion pathogens such as BVDV, BoHV-1, BoHV-5, Serratia marcescens, Campylobacter spp., Brucella spp., Toxoplasma gondii, Neospora caninum, Chlamydophila abortus, Listeria monocytogenes, Leptospira spp., Salmonella enterica Serovar abortusovis, Coxiella burnetii as well as the presence of BTV. The animals originated from 812 farms distributed in 11 different regions of Italy (Table 1). The farms reported reproductive failures following the BT vaccination. Spleen and/or brain from each foetus or malformed offspring was/were collected and stored at −80°C until testing was performed. The isolation or detection methods of the most common abortion-related pathogens of domestic ruminants are listed in Table 2. The presence of BTV was detected as described by the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, OIE, 2008). Briefly, spleen/brain homogenates were inoculated intravascularly in 10-day-old embryonated chicken eggs (ECE). The eggs were incubated in a humid chamber at 32–33 °C and candled daily. Embryos that died between days 2 and 7 were stored at 4°C, and live embryos were killed at day 7. All dead embryos were analysed as two separate pools; the first pool consisted of embryos that died from 2 to 7 days, the other pool consisted of embryos that were killed at day 7. Livers, hearts, spleens, lungs and kidneys of the embryos were pooled and homogenised after homogenization, they were inoculated on confluent baby hamster kidney (BHK21) cells lines. Presence of BTV in cell culture was confirmed by RT-PCR as described by Polci et al. (2007). The BTV serotype involved was identified by virus neutralization according to the methods described in the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals (Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, OIE, 2008). Conversely, whether the BTV-2/9 strains had a vaccine or field origin was determined by using the method described by Elia et al. (2008). The influence of variables like species, period of gestation at the time of vaccination, BTV serotype, foetal organs and the time elapsed between vaccination/abortion on the occurrence of BTV in foetal tissues was also investigated. The average duration of the gestation period for cattle and buffaloes was considered as 39 weeks; while, for sheep and goats, the gestation period was considered to be 22 weeks. According to the period of gestation at the time of vaccination, two groups were considered, the first group included animals vaccinated during the first half of gestation (within 19 weeks for buffalo and cattle, within 11 weeks for sheep and goats); the second group included animals vaccinated during the second half of pregnancy. The maximum duration of viraemia following vaccination with MLV was considered to be 60 days (at least in cattle) (OIE, 2011); dams vaccinated 60 days before mating were also included in the study. For each percentage value, its 95% confidence interval was calculated. The virological data were estimated through a Bayesian approach using the Beta (s + 1, n−s + 1) distribution where s is the total number of positives and n is the total number of tested animals (Sivia, 1996). The peak of the distribution represents the most probable value of the percentage of positive animals, and the wideness gives information about the uncertainty of the estimates due to sample size.

Table 1. Number of herds per region which claimed abortion or fetal death after bluetongue virus (BTV)-2 and BTV-9 vaccination
RegionGoat Sheep Cattle Buffaloes Tot
Abruzzo2971090208
Basilicata113413058
Calabria00303
Campania21428347
Lazio11375512115
Liguria10001
Molise1340510104
Puglia3355371126
Sardegna1103014
Sicilia1916026
Toscana760350102
Total8235635016804
Table 2. Isolation or detection methods used for the most common abortion-related pathogens in domestic ruminants
Abortion-related pathogensIsolation or detection method
BVDVRT-PCR. Baxi et al. (2006)
BoHV-1-BoHV-5PCR. Claus et al. (2005)
Serratia marcescens MacConkey agar
Campylobacter sppCampylobacter blood agar plates
Brucella sppBrucella medium base, TSA
 PCR. Richtzenhain et al. (2002)
Toxoplasma gondii PCR. Hurtado et al. (2001)
Neospora caninum PCR. Masala et al. (2007)
Chlamydophila abortus PCR. Masala et al. (2007)
Listeria monocytogenes Blood agar
Leptospira sppPCR. Richtzenhain et al. (2002)
Coxiella burnetii PCR. Masala et al. (2007)
Salmonella enterica PCR. Masala et al. (2007)
Serovar abortusovis  

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion and Conclusion
  7. Acknowledgements
  8. References

The number of foetuses processed was 1272; 31 (2.4%; 95% CI: 1.7–3.4%) were found to be infected with BTV-2 or BTV-9. Even if no other abortion-related pathogens were detected or isolated from the foetuses (data not shown), the possibility that other undetected pathogens or other factors, such as maternal stress in sheep, might be responsible for the abortions cannot be ruled out. All BTV strains isolated in the foetal tissues were of vaccine origin. No significant association (P > 0.05) was found between species and occurrence of foetal infections. Bluetongue virus was isolated in 24 (3.6%; 95%CI: 2.4–5.3%) ovine, 6 (1.4%; 95% CI: 0.6–3.0%) bovine and 1 (0.6%; 95% CI: 0.2–3.5%) goat foetuses (Fig. 1). Serotype 9 (n = 28) was found in foetal tissues more often (P < 0.05) than serotype 2 (n = 3). The gestation period at the time of vaccination did not affect BTV foetal infection (P > 0.05%). Similar proportions of BTV-infected foetuses were found in animals vaccinated either in the first or in the second half of the gestation period (Table 3). Although the brain was the foetal organ (n = 14) from which BTV was more often isolated, no significant differences (P > 0.05) were found when the number of successful isolations obtained from brain and spleen (n = 6) of the same foetuses was compared. Two BTV-9 vaccine strains were isolated 72 and 67 days after vaccination, respectively, whereas a vaccine strain of BTV-2 was isolated 65 days post-vaccination (Fig. 1). Severe malformations were also observed in 2 newborn calves and 6 lambs born from mothers vaccinated in the first period of pregnancy. Hydranencephaly or hypoplasia of the encephalon with hydrocephalus ex vacuo was the lesions most commonly found (Fig. 2a,b). Although the anamnestic data accounted for vaccine side effects, neither BTV strains nor other pregnancy-related pathogens were isolated or detected from the malformed animals.

image

Figure 1. Number of bluetongue virus (BTV)+ foetuses and time of abortion following vaccination. “X” axis indicates the days elapsed between vaccination and BTV isolation thus giving the survival period of BTV in the foetal tissues. Range of X axis is not on scale. “Y” axis indicates the number of aborted animals from which BTV was isolated.

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image

Figure 2. Malformed animals from dams vaccinated during the first period of pregnancy. Lamb (a) and calf (b) with hydranencephaly.

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Table 3. Relationship between numbers of aborted fetuses tested for BTVs by VI and the gestation period when BTV vaccination occurred
SpeciesVaccination periodVI
PositiveNegative
  1. BTV, Bluetongue virus; VI, virus isolation; dd, days.

GoatWithin 60 dd from mating014
Up to the 11th week081
After the 11th week159
SheepWithin 60 dd from mating0103
Up to the 11th week10275
After the 11th week14261
CattleWithin 60 dd from mating014
Up to the 19th week3184
After the 19th week3225
BuffaloesWithin 60 dd from mating01
Up to the 19th week07
After the 19th week09

Discussion and Conclusion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion and Conclusion
  7. Acknowledgements
  8. References

In Italy, the first evidence of BT infection was recorded in Sardinia on August and in Calabria on November 2000, respectively. BTV-2 and BTV-9 were the serotypes involved (Caporale and Giovannini, 2010). Italian authorities 3 years later implemented a vaccination campaign according to the geographic distribution of the incurring BTV serotypes and the availability of appropriate vaccines; this campaign was carried out to reduce direct losses because of disease and indirect losses because of the trade ban caused by virus circulation, The campaign used the BTV-2 and BTV-9 MLVs manufactured by OBP and involved all domestic ruminants. The vaccines that had been in use in South Africa for over forty years were the only commercial products available at that time. The concerns in the use of MLVs were related to the possibility of side effects in the vaccinated animal, due to the potential of the MLV to replicate in the vaccinated organism at titres capable of infecting the vector and thus spreading in the environment, but also on the possibility that MLV might cross the placental barrier, infect foetuses and cause abortion, malformation, stillbirth or neonatal mortality (Flanagan and Johnson, 1995).

Both BTV-2 and BTV-9 MLVs were attenuated in a similar manner. The BTV-2 strain selected by OBP for attenuation was passaged 50 times in embryonated chicken eggs, plaque selected three times and passaged twice in BHK21 cells, whereas the BTV-9 strain was passaged 70 times in eggs followed by three small plaque selection and six further passages in BHK21 cells (Savini et al., 2008). These MLVs, originally produced for sheep, were also shown to be safe in cattle. No adverse effects on reproduction (abortion or teratogen defects) were observed in cattle immunized with the monovalent BTV-2 vaccine, or with a combination of BTV-2 and BTV-9 monovalent vaccines, either in controlled or in field conditions (Lucifora et al., 2004; Monaco et al., 2004). More than 10 million animals were vaccinated with the monovalent BTV-2 or the combination of BTV-2/BTV-9 monovalent vaccines during the 2003 Italian vaccination campaign. All adverse effects observed on foetuses following BTV vaccination were investigated in this article. The aborted foetuses tested were 1272; 31 were found to be infected with BTV-2 or BTV-9 vaccine strains. This was the first time that these two serotypes were proven to be able to cross the placental barrier. According to this survey, BTV-9 MLV crossed the placental barrier more often than BTV-2 MLV. BTV-9 MLV was attenuated through a higher number of in vitro passages, thus supporting the hypothesis that a greater in vitro adaptation may enhance the capability of crossing the placental barrier (Johnson et al., 1992; MacLachlan et al., 2000). This BTV-9 MLV crossing capability might derive from a different viral quasi-species composition of the vaccine stock after the egg/cells in vitro passages and/or from an in vivo selection in favour of those viral subpopulations with a strong aptitude to cross the placental barrier. Both MLV strains appear to invade the foetuses indiscriminately; both spleen and brain were infected, and abortions occurred equally in cattle, sheep and goats without any significant difference. However, because of the low number of cases analysed, it is hard to confirm whether this result could be really attributed to the lack of a species effect or alternatively to the low number of reported cases. The laboratory findings indicated that the gestation period at the time of vaccination did not influence the occurrence of abortions in which BTV was isolated. As described by MacLachlan et al. (2000), infection during the earlier stages of pregnancy caused more severe malformations. All the malformed foetuses observed in this study came from dams vaccinated in the very early period of pregnancy. The dams in some cases were vaccinated even a few days before mating. The above phenomenon may be related to the amount of MLVs that cross the dam/foetus placental barrier during the viraemia. Hence, it cannot be excluded that dams vaccinated before mating may preserve enough levels of viraemia for foetus infection. Further experiments are necessary in order to elucidate the levels of viraemia that might be potentially dangerous for the foetus and the capability of BTV to cross the placental barrier in the earliest days of conception. Interestingly, all malformed animals turned out to be BT negative by VI and bio-molecular techniques, phenomenon already described in the past (MacLachlan and Osburn, 1983). After an early infection, it seems that foetuses are able to get free of BTV by the time of delivery which might have been responsible for an underestimation of the BTV MLV foetal infections. Infectious BTV-2 and BTV-9 vaccine strains were detected in three ovine foetuses, 65–72 days after vaccination, a period far longer than the expected BTV-infectious period known for this species; this prolonged infectious period might allow BTV to survive winter climates. The survival characteristic of BT MLVs might play a relevant role in the epidemiology of BT MLV strains. The fact that BTVs were found in a very low percentage of aborted animals implied that the epidemiological role played by this phenomenon might be not significant. During the BTV vaccination campaign, 312 of 87245 holdings on which vaccination was performed reported adverse effects. They represent the 0.16% of vaccinated cattle herds and 0.50% of vaccinated small ruminant flocks. Vaccine strains confirmed by the laboratory were present in 47 holdings (0.01% of vaccinated cattle herds and 0.09% of vaccinated sheep and goat flocks). As discussed by Caporale and Giovannini (2010), comparable rates of adverse effects (0.01%) following vaccination were found in the United States by the Vaccine Adverse Event Reporting System (VAERS) for 27 different types of human vaccines between 1991 and 2001.

In conclusion, this study evidenced a tight and clear connection between abortion/teratogen effects and vaccination with BTV-2 and BTV-9 MLVs. Even though more than a thousand abortions occurred following the BTV vaccination campaign, they only represent a small percentage of the total vaccinated animals. Considering that in just a small percentage of them BTV was detected, it could be concluded that the vaccination campaigns with BTV2/9 MLVs in Italy was, in terms of cost/benefit analysis, more than satisfactory. Abortion and teratogen effects represent a big concern for the livestock industry. Recent outbreaks of Schmallenberg virus (SBV) in Northern Europe warrant a European common surveillance programme for livestock abortion and malformations. The understanding of the mechanisms that permit several pathogens, including SBV and BTV, to cross the placental barrier, becomes crucial. Full genome sequence analyses of the vaccine strains isolated in the foetuses might elucidate the mechanisms responsible for the longer survival of MLV strains in the foetal organs as well as the higher capability of BTV-9 to cross the placental barrier. The identification of the point mutations in the BTV gene segments, which are likely involved in the vertical transmission, might help to promptly detect novel BTV field strains capable of crossing the placental barrier and prevent possible reassortment.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion and Conclusion
  7. Acknowledgements
  8. References

We thank Dr. Sandro Santarelli for editing the pictures of the manuscript and Alfreda Tonelli for the English review.

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  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion and Conclusion
  7. Acknowledgements
  8. References
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