Canine Herpesvirus During Pregnancy and Non-Pregnant Luteal Phase

Authors


Author's address (for correspondence): B Strom Holst, Department of Clinical Sciences, PO Box 7054, Swedish University of Agricultural Sciences, SE-750 07 Uppsala, Sweden. E-mail: Bodil.Strom-Holst@slu.se

Contents

Canine herpesvirus (CHV) is a widespread infection among dogs that typically get latently infected after exposure and can reactivate the infection after stress. The aim of the present study was to study the effects of latent CHV infection during pregnancy on pregnancy outcome, and to study if there are signs of genital viral reactivation during pregnancy or during non-pregnant luteal phase. Twelve mated bitches and eight control bitches were followed and sampled regularly during pregnancy or non-pregnant luteal phase. Blood samples were taken for antibody analysis and vaginal swabs for real-time PCR analysis. Three of the pregnant bitches were vaccinated against CHV during pregnancy. All bitches had antibodies to CHV. Two pregnant bitches that were not vaccinated had a twofold or larger increase in CHV titre, with no negative effects detected on pregnancy. Higher titres were not associated with smaller litters or with vaccination. There was no consistent variation in antibody titres due to pregnancy or non-pregnant luteal phase. Vaginal excretion of CHV was not detected from any of the bitches.

Introduction

Canine herpesvirus (CHV) is common in Europe, and seroprevalences of 40–90% have been reported (Reading and Field 1999; Ronsse et al. 2002). Although CHV is a widespread infection among dogs, the consequences of the infection largely remain unknown. It is well known that CHV causes mortality in neonatal pups, especially when the bitch is infected late in pregnancy. As a large proportion of the canine population is latently infected, they can potentially reactivate the infection during for instance oestrus, pregnancy, lactation or other stressful situations. Via different mechanisms involved, both 17-beta estradiol and medroxyprogesterone acetate have been shown to promote herpes simplex virus type 1 reactivation in mice (Cherpes et al. 2008; Vicetti Miguel et al. 2010). Appearance of vulvovaginal papules in bitches, related to CHV infection, has been associated with stage of the oestrous cycle (Poste and King 1971). Thus, there appears to be an association between viral reactivation and circulating reproductive hormone concentrations.

Clinical effects related to reactivation of a latent CHV infection have not been clarified, although reproductive disturbances and lower pup weights at birth have been suggested (Poulet et al. 2001). In kennels with many infected bitches, a variety of reproductive disorders during pregnancy have been described, such as resorptions, mummifications and abortions (Ronsse et al. 2005). The aim of the present study was to study the effects of latent CHV infection during pregnancy on pregnancy outcome, and to study if there are signs of genital viral reactivation during pregnancy or during non-pregnant luteal phase.

Materials and Methods

Twenty bitches were originally included in the study; 12 case bitches that were mated or inseminated and eight control bitches. The case bitches were two Labrador retrievers and two German shepherds, and one bitch each of the following breeds: Golden retriever, Stabyhoun, Chow Chow, Bernese Mountain dogs, Leonberger, Polski Owczarek Nizinny (PON), Schapendoes and Shetland sheepdog. The mean age of the case bitches was 4.1 years. All bitches had contact with other dogs, and no bitch had a history of previous pregnancy losses due to CHV. Seven of the bitches were nulliparous, and five had given birth to one or more litters. Three bitches (the stabyhoun, the PON and the Shetland sheepdog) were vaccinated against CHV (Eurican Herpes 205, Merial) during the study. Descriptive data from these bitches are shown, but they were excluded from further calculations: Details of the case bitches are given in Table 1. The control bitches were five beagles and one mixed breed living in a colony of research dogs, and two privately owned dogs: one German shepherd and one Rottweiler. Their mean age was 3.4 years. Samples were collected from the bitches at optimal mating time: a fully cornified vaginal smear and progesterone concentrations above 30 nm (Day 1) and then on day 8, 15, 22, 29 and 43. Control bitches were additionally sampled on days 57 and 71.

Table 1. Bitches with offspring mortality. The canine herpesvirus (CHV) titres on day 1 and day 43 of the study are shown, as well as litter size and offspring mortality up to 8 weeks of age
BreedKept in house (H) or Kennel (K)CHV titre day 1CHV titre day 43Embryonic death detectedPup death; diagnosis established at autopsy (DA)Litter size
Labrador retrieverK16016001 stillborn, no specific DA1
German shepherdH16016011 stillborn, no specific DA1
Golden retrieverK160160009
German shepherdH16064002 stillborn; DA: trauma10
Chow chowH32064002 stillborn no specific DA, and one dead at day 3; no autopsy performed10
Bernese mountain dogH64064002 stillborn; no autopsy performed13
LeonbergerH640160009
SchapendoesH16032001 dead at 8th week; DA: Patent ductus arteriosus4
Labrador retrieverH80320006
Shetland sheepdogH320160 (vaccinated)103
StabyhounH320160 (vaccinated)001
PONH320160 (vaccinated)006

Blood samples were taken from the cephalic vein into test tubes without additives. After centrifugation, serum was stored at −20°C before analysis of antibodies to CHV. In addition, a vaginal swab was taken and stored frozen at −20°C until analysed by real-time PCR for CHV. On days 22 and 29, pregnancy diagnosis by ultrasonography (Acuson Antares, VFX 13–5 MHz linear probe; Siemens Medical Solutions, Mountain Dew, CA, USA) was performed. Post-mortem investigation was offered for all stillborn pups and pups that died before 8 weeks of age, with special emphasis on CHV including real-time PCR analysis of organs when relevant.

Antibody analysis

For antibody analysis, an immunoperoxidase monolayer assay (IPMA) was used (Wellenberg et al. 1999) as modified by (Krogenaes et al. 2012). All samples were run at the same time. As negative control, serum from specific pathogen-free dogs was used, and as positive control dog serum with a titre ≥160 from previous analyses was used. Titres <80 were defined as negative. Positive antibody titres were categorized as 80 = weakly positive, 160 and 320 = moderately positive, 640 and 1280 = strongly positive (Krogenaes et al. 2012).

Real-time PCR analysis

Extraction of DNA from vaginal swab samples was performed by first cutting the swabs into test tubes and shaking them in 850-μl TE buffer and then using magnetic bead capture (Boom silica) of nucleic acids using the Nordiag Vet Viral NA kit in a Magnetic Biosolutions Magnatrix 8000+ extraction robot.

For PCR analysis of CHV-1, an in-house developed real-time PCR assay targeting the glycoprotein B (gB) gene was used. The positive control used was an isolate from organ material. The primers and probe were 5′-CGAGCAGAGTTAGAAGATACAGGA-3′, 5′-TTGCAGCACCCAAAACAACTTT-3′, and 6-FAM-CCCGCTCCAACATCTCCAAGTCCT-BHQ-1. The primers and probe were compared with published sequences in Nucleotide Basic Local Alignment search Tool (blast). They were found to match 100% with the three published gB sequences, whereas no closely related sequences would give rise to a signal. Reactions were run using the Agpath ID one-step RT-PCR kit (Life Technologies, Carlsbad, CA, USA) as described in the kit manual, but in a smaller volume: 13-μl mastermix and 2-μl template in a total volume of 15 μl. Samples were run in a 7500 Applied Biosystems real-time instrument (Life Technologies, Carlsbad, CA, USA). Samples producing typical reaction curves and Ct < 40 were considered positive.

Statistical analysis

Minitab statistical software was used for statistical analyses. For groupwise comparisons (antibody titres for bitches with litters of 1–3 pups compared with bitches with larger litters), the Kruskal–Wallis test was used. In the graph of antibody titres, results are presented as mean ± 95% confidence intervals (statistical significance represented by non-overlapping CIs).

Ethical consideration

The study was approved by the Uppsala Ethical Committee of Animal Experimentation (C23/9) and the Swedish Board of Agriculture (31-1365/09).

Results

Reproductive performance and offspring mortality

All bitches that were mated or inseminated conceived and delivered pups. In total, 74 pups were born, with a mean litter size of 6.2 pups. Embryonic mortality was detected by ultrasonography in two bitches, one of which was vaccinated. From a total of six mothers, eight pups were stillborn and another two pups died before 8 weeks of age. Seven of the ten pups were submitted to post-mortem analysis. For three pups, the dog owners were convinced of another cause for death than CHV and therefore did not send the pups for autopsy. In the pups that were autopsied, no signs of CHV infection were detected. Details on bitches and offspring mortality are given in Table 1.

CHV serology and PCR results

All bitches had antibodies to CHV. All three case bitches that were vaccinated against CHV had antibodies to CHV before vaccination. Two of the vaccinated bitches had the same titre 2 weeks after vaccination and the last vaccinated bitch, had an increase of one dilution step, from 320 to 640.

Of the case bitches, one had low, nine had moderate and two had high titres on day 1. Of the control bitches, six had moderate and two had high titres on day 1. During the study period, antibody titres varied by two or more dilution steps in five mated and two control bitches. An increase by two dilution steps in two consecutive analyses was only seen in one of the mated bitches, from 80 to 320 on days 1–8 and days 22–29. This was a Labrador retriever with no embryonic or pup mortality detected and a litter size of 6. A German shepherd had an increase between several samplings (from 80 day 8, 160 day 15, 320 day 22 to 640 day 29), with 10 pups born and two neonatal deaths due to trauma. One bitch had a steady decrease in antibody titre during the study, from 640 to 160, with a litter of nine pups and no offspring mortality.

No consistent changes in antibody titres were seen that could be attributed to pregnancy or luteal phase, and based on overlapping confidence intervals, there were no significant differences between pregnant and non-pregnant bitches (Fig. 1). When antibody titres on day 1 and day 43 were compared for bitches with litters of 1–3 pups compared with bitches with larger litters, there was no significant difference in antibody titre between the groups for either day. The two bitches with embryonic mortality were weakly to moderately positive for antibodies to CHV. CHV could not be detected from any vaginal sample.

Figure 1.

Variations in antibody titres during pregnant (case bitches, excluding the three vaccinated bitches) and non-pregnant (control bitches) luteal phase. Mean values and 95% confidence intervals are shown

Discussion

Canine herpesvirus is a common infection in Europe, with a high seroprevalence described in several countries (Krogenaes et al. 2012). This is supported by the fact that all bitches in the present study had antibodies to CHV. A major reproductive disturbance linked to infection with CHV is death of pups younger than 3 weeks, but other reproductive problems may occur. In a study from Finland, CHV antibody titres were higher in kennels with reproductive problems such as low conception rates, embryonic losses, abortions and stillborn pups (Dahlbom et al. 2009). In another study, bitches vaccinated against CHV gave birth to significantly heavier pups than unvaccinated bitches (Poulet et al. 2001). Bitches latently infected by experimental intraocular infection with CHV have been shown to reactivate the infection, showing ocular signs, ocular viral shedding and a fourfold elevation in CHV antibody titre, after administration of prednisolone (Ledbetter et al. 2009).

In the present study, all bitches had antibodies to CHV. Bitches with twofold or larger increases in CHV titre during the study had no detected negative effects on pregnancy. Embryonic death was detected by ultrasonography in two of the 12 pregnant bitches but was not related to high or increasing CHV titres. Higher titres to CHV were not associated with smaller litters. The fact that no signs of CHV were detected in the autopsied pups implies that CHV was not involved in these losses.

In a previous study, a significant decrease in antibody titre was seen in early dioestrus (Ronsse et al. 2004). In the present study, there was no consistent variation in antibody titres due to pregnancy or non-pregnant luteal phase. Latent infection with CHV has been detected in the lumbo-sacral ganglia (Burr et al. 1996), and after treatment with prednisolone, CHV has been detected from vaginal samples (Okuda et al. 1993a), although not as frequently as from samples from the upper respiratory tract (Okuda et al. 1993b). Reproductive disturbances due to reactivation of a latent CHV infection in a bitch with a non-affected general condition would more likely be due to reactivation of virus in the lumbo-sacral ganglia than in, for instance, the tonsils. A reactivation of the virus in the genital tract could possibly also be detected from vaginal samples. No vaginal excretion of CHV was detected in the present study.

To conclude, the seroprevalence of CHV in the general dog population is widespread. This current study, on a limited number of bitches, could not demonstrate any negative effects on pregnancy, litter size, or pup mortality that could be related to a latent CHV infection. Pregnancy alone does not seem to cause reactivation of the infection when management is good, and other stressing factors are absent.

Acknowledgement

The present study is a part of the BITCH project, funded by the Agria Insurance and Swedish Kennel Club Research Foundation.

Conflicts of interest

None of the authors have any conflicts of interest to declare.

Ancillary