Testicular Degeneration and Infertility following Arbovirus Infection.

Arboviruses can cause a variety of clinical signs, including febrile illness, arthritis, encephalitis, and hemorrhagic fever. The recent Zika epidemic highlighted the possibility that arboviruses may also negatively affect the male reproductive tract. In this study, we focused on bluetongue virus (BTV), the causative agent of bluetongue and one of the major arboviruses of ruminants. We show that rams that recovered from bluetongue displayed signs of testicular degeneration and azoospermia up to 100 days after the initial infection. Importantly, testicular degeneration was induced in rams experimentally infected with either a high (BTV-1IT2006)- or a low (BTV-1IT2013)-virulence strain of BTV. Rams infected with the low-virulence BTV strain displayed testicular lesions in the absence of other major clinical signs. Testicular lesions in BTV-infected rams were due to viral replication in the endothelial cells of the peritubular areas of the testes, resulting in stimulation of a type I interferon response, reduction of testosterone biosynthesis by Leydig cells and destruction of Sertoli cells and the blood-testis barrier in more severe cases. Hence, BTV induces testicular degeneration and disruption of spermatogenesis by replicating solely in the endothelial cells of the peritubular areas unlike other gonadotropic viruses. This study shows that a naturally occurring arboviral disease can cause testicular degeneration and affect male fertility at least temporarily.IMPORTANCE During the recent Zika epidemic, it has become apparent that arboviruses could potentially cause reproductive health problems in male patients. Little is known regarding the effects that arboviruses have on the male reproductive tract. Here, we studied bluetongue virus (BTV), an arbovirus of ruminants, and its effects on the testes of rams. We show that BTV was able to induce testicular degeneration in naturally and experimentally infected rams. Testicular degeneration was caused by BTV replication in the endothelial cells of the peritubular area surrounding the seminiferous tubules (the functional unit of the testes) and was associated with a localized type I interferon response, destruction of the cells supporting the developing germinal cells (Sertoli cells), and reduction of testosterone synthesis. As a result of BTV infection, rams became azoospermic. This study highlights that problems in the male reproductive tract caused by arboviruses could be more common than previously thought.

Quantification of the increase in the frequency of early calving associated with late exposure to bluetongue virus serotype 8 in dairy cows: implications for syndromic surveillance.

A recent study evaluating whether reproductive data could be used for syndromic surveillance found an increased frequency of early calving (calving occurring a few days earlier than expected) in areas exposed to the Bluetongue virus serotype 8 (BTV-8) in northern Europe. A high proportion of herds infected during the 2006-2009 European outbreak were not reported through the surveillance system. The objectives of this study were (1) to quantify the increase in the frequency of early calving associated with the exposure to BTV-8 in late gestation and (2) to determine whether this association could be found in populations exposed to BTV-8 but without reported clinical signs. Increases in frequency of early calving were quantified for cows in herds located in the 2007 outbreak area in France, reported or not as cases. Increases were detected for cows in both categories of herds with a larger effect in herds reported after clinical signs. Moreover, the largest effect was found for exposures occurring during the latest stage of pregnancy, suggesting that BTV infection could trigger calving in cows in late gestation, a few days earlier than expected. This is the first study quantifying the association between a viral infection and a shortened pregnancy length (still within a normal range). The high magnitude of the increase in frequency of early calving, their occurrence in herds from infected areas but not reported, and the short time interval between exposure and the occurrence of the event confirm the interest of using early calving as an indicator for syndromic surveillance.

Can routinely recorded reproductive events be used as indicators of disease emergence in dairy cattle? An evaluation of 5 indicators during the emergence of bluetongue virus in France in 2007 and 2008.

In response to increasing risks of emerging infectious diseases, syndromic surveillance can be a suitable approach to detect outbreaks of such diseases across a large territory in an early phase. To implement a syndromic surveillance system, the primary challenge is to find appropriate health-related data. The objective of this study was to evaluate whether routinely collected dates of reproductive events in dairy cattle could be used to build indicators of health anomalies for syndromic surveillance. The evaluation was performed on data collected in France between 2003 and 2009. First, a set of 5 indicators was proposed to assess several types of reproductive disorders. For each indicator, the demographic coverage over the total number of cattle at risk was analyzed in time and space. Second, the ability to detect an emerging disease in an early phase was retrospectively evaluated during epidemics of bluetongue serotypes 1 and 8 (BTV-1, BTV-8) in France in 2007 and 2008. Reproductive indicators were analyzed weekly during these epidemics for each indicator in each infected French district (16 in 2007 and 50 in 2008 out of 94 districts). The indicators were able to detect the BTV epidemics despite their low demographic coverage on a weekly basis relatively to total number of cattle (median=1.21%; range=0-11.7%). Four indicators related to abortions, late embryonic death, and short gestations were abnormally elevated during both BTV epidemics. Median times to abnormal elevations in these indicators were 20 to 71 d after the first notification of clinical signs of BTV by veterinarians. These results demonstrate that reproduction data can be used as indicators of disease emergences, whereas in the specific case of these BTV epidemics, detection via these indicators was later than clinical detection by veterinarians. The emergence of bluetongue in 2007 in France was associated with gestations that were a few days shorter than expected. A short gestation indicator underwent high elevations relative to prior random fluctuations and was the earliest (out of the 4 indicators) to show abnormal elevations, making it possible to detect this emergence.

Isolation of bluetongue virus serotype 1 from aborted goat fetuses.

Abortions and stillbirths were noticed in pregnant goats on a farm in the state of Gujarat, India. About 50% of the pregnant goats aborted or gave birth to dead kids. Bluetongue virus (BTV) antibody in the sera of affected goats was detected using a competitive enzyme-linked immunosorbent assay (ELISA). Viral antigen in the blood of these goats and in the aborted fetal spleens was detected using a sandwich ELISA. Two viruses (SKN-9, SKN-10) were isolated in cell culture from aborted fetal spleens and were confirmed as Orbivirus by demonstration of ten bands in RNA polyacrylamide gel electrophoresis and identified as BTV-1 by sequencing of the VP2 gene. Sequence analyses revealed thatthese isolates were very closely related to a BTV-1 (strain SKN-8) isolated from Culicoides vectors captured on the same farm one month after the occurrence of abortion. Isolation of BTV-1 from fetuses is probably evidence of transplacental transmission of the wild-type strain, because attenuated or laboratory-adapted BTV-1 strains have never been used in this region. This may have important implications in the epidemiology of bluetongue, considering the presence of many BTV serotypes in India.

Decrease in milk yield associated with exposure to bluetongue virus serotype 8 in cattle herds.

Decreased milk yield and reduced fertility are the primary consequences of infection by bluetongue virus serotype 8 (BTV-8). These effects must be quantified to fully assess the economic benefit of vaccination. This can be estimated by measuring the effect of BTV-8 exposure on milk yield and fertility for all cows belonging to an infected herd. The objectives of this study were (1) to quantify the mean effect of exposure to BTV-8 on milk yield following natural challenge for cows in herds previously naïve, (2) to determine the duration of reduced milk yield before and after the date disease was first detected in the herd to estimate the cumulative loss of milk yield during this period, and (3) to evaluate the influence of the proportion of infected neighboring herds on the reduction in milk yield following exposure to BTV-8. The effects of exposure to BTV-8 during the French outbreak of 2007 were assessed using mixed linear models, which allow adjustment for factors known to influence milk yield. Exposure to BTV-8 was associated with a sharp decrease in milk yield over a period of 6 mo (2 mo before to 4 mo after the reported date of disease detection in the herd). The cumulative loss of milk yield was more than 3% of annual production. The relatively earlier reduction in milk yield in infected herds detected later in the outbreak period suggests that detection of clinical signs was delayed in these herds. Finally, the greatest decrease in milk yield was observed in herds detected early during the outbreak period and located in areas with the highest disease incidence. This may be due to a greater within-herd incidence or to a greater amount of virus injected by midges to individual cows in these herds.

Quantification and at-risk period of decreased fertility associated with exposure to bluetongue virus serotype 8 in naïve dairy herds.

The detrimental effect of bluetongue virus serotype 8 (BTV-8) on fertility was quantified in seroconverting cows. Although the effect on individual cows provides information regarding the potential biological burden of infection, losses at a herd level are also dependent on the proportion of infected cows within the herd. The objectives of this study were to quantify the average effect of BTV-8 exposure in field conditions on the fertility of dairy cows in previously naïve herds, and to determine the at-risk period of decreased fertility related to the date of detection of the disease in the herd. The effect of BTV-8 exposure on fertility was assessed using the 90-d-return-to-service rates after the first artificial insemination (AI) calculated for cows in exposed herds (during the 2007 epizootic in France) and compared with that for cows in unexposed herds. Only herds with a confirmed detection that were reported after clinical suspicion were included. To determine the at-risk period of decreased fertility, variations of fertility in exposed herds were quantified according to the time interval between the date of AI for individual cows and the date that disease was detected in the herd. Survival analyses were used to assess the risk of decreased fertility associated with BTV-8 exposure, adjusting for the main factors known to influence fertility. The episode at risk for decreased fertility depended on the month of disease detection in the herd. For herds detected early in the epizootic, fertility was decreased for cows inseminated from 1 mo before to 1 mo after the date of disease detection in the herd. Depending on time interval between the date of AI of cows and the date of detection in the herd, the increase of return-to-service rate associated with BTV-8 exposure varied from 8 to 21 percentage points of 90-d return to service. The episode of decreased fertility is likely due to a combination of the effect of the infection at different stages of conception and early pregnancy and the delayed exposure of cows due to the spreading of the virus within herds. For herds detected during the second half of the epizootic, fertility was decreased for cows inseminated more than 2 mo before detection, which suggests a delay in the detection of clinical signs following virus introduction in the herd. No correlation was observed between the effect of BTV-8 exposure on fertility and the incidence of BTV-8 in the local geographical area. Given the duration of the period that cows were at risk for decreased fertility and the magnitude of the effect, the average BTV-8 exposure in naïve herds led to major losses.

[Investigation of abortions and other animal health problems in relation to vaccination against Bluetongue virus in 2009]. / Abklärung von Aborten und anderen tiergesundheitlichen Problemen beim Rind im Zusammenhang mit der Blauzungenvirus-Impfung 2009.

By the distribution of a questionnaire between all Swiss cattle practitioners it was possible to investigate abortions and other animal health problems related to Bluetongue vaccination 2009. The questionnaire helped to obtain plausibility and timely relation of the reported disorders. 58 abortions in cattle and different herd health problems could be examined. Because there is no possibility to show that a vaccination itself leads to an abortion the results of proven causes of abortions prior and after Bluetongue vaccination were compared regarding their diagnosis. Due to the fact that diagnosis and solving rate of abortions did not differ before and after vaccination, the vaccination itself cannot be responsible for the abortions. Evaluation of different herd health problems showed that Bluetongue vaccination was not responsible for these disorders which often existed already prior to vaccination. Herd health problems generally have multifactorial causes what makes it difficult to asses the effect of Bluetongue vaccination in some cases.

Identification and characterization of epizootic hemorrhagic disease virus serotype 6 in cattle co-infected with bluetongue virus in Trinidad, West Indies.

Epizootic hemorrhagic disease virus (EHDV) is an economically important virus that can cause severe clinical disease in deer and to a lesser extent cattle. This study set out to determine and characterize which EHDV serotypes were circulating in Trinidad. Serum and whole blood samples were collected monthly for six months from a cohort of cattle imported to Trinidad from the USA. Results revealed that all the cattle seroconverted to EHDV within six months of their arrival, with EHDV RNA being detected in the samples just prior to antibodies, as expected. Serotyping assays revealed that a single serotype (EHDV-6) was circulating in the cattle. Sequencing of the surface viral protein (VP2) of EHDV-6, followed by phylogenetic analysis, revealed that the Trinidad EHDV-6 strain was closely related to EHDV-6 viruses found in Guadeloupe (2010), Martinique (2010) and USA (2006), with 96-97.2% nucleotide identity. The Trinidad EHDV-6 VP-2 shared 97.2% identity with the Australian EHDV-6 prototype strain, classifying it within the eastern topotype clade. Bayesian coalescent analysis support Australia as the most probable source for the EHDV-6 VP2 sequences in the Americas and Caribbean region and suggests that the they diverged from the Australian prototype strain around 1966 (95% HPD 1941-1979).

[Bluetongue virus serotype 8 (BTV-8)-associated brain malformations in two calves]. / Blauzungenvirus Serotyp 8 (BTV-8)-assoziierte Gehirnmissbildungen bei zwei Kälbern.

Congenital brain malformations such as hydranencephaly as well as internal and external hydrocephalus combined with porencephaly were diagnosed in two calves which were born in spring 2008. In both calves bluetongue virus was detected by real-time PCR. Teratogenic pestiviruses were not found by serological, molecular or immunohistological methods. A causal relationship between the malformations and the bluetongue serotype 8 epidemic in 2007 has to be considered.

Teratogenic bluetongue and related orbivirus infections in pregnant ruminant livestock: timing and pathogen genetics are critical.

Congenital infections of domestic animals with viruses in several families, including Bunyaviridae, Flaviridae, Parvoviridae, and Reoviridae, are the cause of naturally occurring teratogenic central nervous system and/or musculoskeletal defects (arthrogryposis) in domestic animals. Congenital infections of ruminant livestock with bluetongue virus (BTV) and some related members of the genus Orbivirus (family Reoviridae) have clearly shown the critical role of gestational age at infection in determining outcome. Specifically, fetuses infected prior to mid-gestation that survive congenital BTV infection are born with cavitating central nervous system defects that range from severe hydranencephaly to cerebral cysts (porencephaly). Generally, the younger the fetus (in terms of gestational age) at infection, the more severe the teratogenic lesion at birth. Age-dependent virus infection and destruction of neuronal and/or glial cell precursors that populate the developing central nervous system are responsible for these naturally occurring virus-induced congenital defects of animals, thus lesions are most severe when progenitor cells are infected prior to their normal migration during embryogenesis. Whereas congenital infection is characteristic of certain BTV strains, notably live-attenuated (modified-live) vaccine viruses that have been passaged in embryonating eggs, transplacental transmission is not characteristic of many field strains of the virus and much remains to be determined regarding the genetic determinants of transplacental transmission of individual virus strains.

Syndromic surveillance of abortions in beef cattle based on the prospective analysis of spatio-temporal variations of calvings.

Our objective was to study the ability of a syndromic surveillance system to identify spatio-temporal clusters of drops in the number of calvings among beef cows during the Bluetongue epizootic of 2007 and 2008, based on calving seasons. France was partitioned into 300 iso-populated units, i.e. units with quite the same number of beef cattle. Only 1% of clusters were unlikely to be related to Bluetongue. Clusters were detected during the calving season of primary infection by Bluetongue in 28% (n = 23) of the units first infected in 2007, and in 87% (n = 184) of the units first infected in 2008. In units in which a first cluster was detected over their calving season of primary infection, Bluetongue was detected more rapidly after the start of the calving season and its prevalence was higher than in other units. We believe that this type of syndromic surveillance system could improve the surveillance of abortive events in French cattle. Besides, our approach should be used to develop syndromic surveillance systems for other diseases and purposes, and in other settings, to avoid "false" alarms due to isolated events and homogenize the ability to detect abnormal variations of indicator amongst iso-populated units.

Bluetongue and equine viral arteritis viruses as models of virus-induced fetal injury and abortion.

A number of viruses have the capacity to cross the placenta and infect the fetus to cause, among other potential outcomes, developmental defects (teratogenesis), fetal death and abortion. Bluetongue virus (BTV) infection of fetal ruminants provides an excellent model for the study of virus-induced teratogenesis. This model has shown that only viruses modified by passage in cell culture, such as modified live virus vaccine strains, readily cross the ruminant placenta, and that the timing of fetal infection determines the outcome. Thus, cerebral malformations only occur after fetal infection at critical stages during development and the precise timing of fetal BTV infection determines the severity of the malformation present at birth. Fetal BTV infection also can result in fetal death, followed by abortion or resorption, growth retardation, or no obvious abnormalities, depending on age of the conceptus at infection. Equine arteritis virus (EAV) infection of the equine fetus causes fetal death and abortion but not teratogenesis. These two fetal viral infections are useful not only for the study of teratogenesis and fetal disease, but also to further characterize and compare the complex process that is responsible for normal induction of parturition in ruminants and horses.

Bluetongue virus in pregnant elk and their calves.

Two pregnant North American elk (Cervus canadensis), in the 3rd and 4th months of gestation, were inoculated with bluetongue (BT) virus (BTV) serotype 11. The virus was not isolated from the blood of the cows beyond postinoculation day (PID) 8, but was isolated from bone marrow and spleen samples obtained at necropsy on PID 190. Although neither cow had overt clinical signs of BT infection, fluctuations in specific neutralizing BTV antibody titers indicated viral replication. However, in 2 attempts, BTV was not recovered biologically via bites of colonized Culicoides variipennis (biting gnats) with subsequent transmission of the BTV to sheep. Bluetongue virus was isolated from the elk calves at birth and before they nursed. These calves remained latently infected, and BTV was transmitted from each calf to sheep by bites of the biting gnats. Most of the BTV biological recovery attempts resulted in suspicious BT clinical responses in sheep, but without viral isolation. However, after challenge exposure with the homologous virus, 5 of 7 recipient sheep bitten by the gnats reacted with an intensified BT clinical response that indicated viral sensitization. One calf was born weak, never attained a healthy appearance, was latently infected with BTV, and had fluctuating BTV neutralizing antibody titers. The other calf was in apparently good health, was latently infected with BTV, and was immunologically tolerant to BTV.

Experimentally induced bluetongue virus infection in white-tailed deer: ultrastructural findings.

White-tailed deer (Odocoileus virginianus) were inoculated with bluetongue virus serotype 17 and sequentially euthanatized during infection. Ultrastructural changes in the microvasculature of tongue, buccal mucosa, heart, and pulmonary artery, platelets, and bone marrow were evaluated. Bluetongue virus was found in endothelial cells of the microvasculature by postinoculation day 4. Viral replication was associated with the development of viral matrices, viral-associated macrotubules, and aggregates of mature viral particles in the cytoplasm of infected cells. Viral infection of pericytes and vascular smooth muscle cells developed subsequent to endothelial cell infection. Viral infection was associated with striking changes in the endothelial lining of the microvasculature by postinoculation day 4. Endothelial cell degeneration and necrosis, which resulted in denudation of the endothelial lining, and endothelial cell hypertrophy frequently were observed. Thrombosis, hemorrhage, and vessel rupture developed subsequent to endothelial damage. Bluetongue virus neither infected nor directly damaged platelets or bone marrow cells. It was concluded that viral-induced endothelial damage is the primary triggering mechanism for disseminated intravascular coagulation in bluetongue virus infection. Vascular damage coupled with the development of disseminated intravascular coagulation is responsible for the hemorrhagic diathesis, which is characteristic of bluetongue virus infection in white-tailed deer.

Experimentally induced bluetongue virus infection in white-tailed deer: coagulation, clinical pathologic, and gross pathologic changes.

Ten yearling white-tailed deer (Odocoileus virginianus) were inoculated with bluetongue virus serotype 17. Two yearling white-tailed deer were inoculated with sonicated heparinized noninfected blood and served as controls. Clinical signs of bluetongue virus infection included increased rectal temperature, erythema, facial edema, coronitis, and stomatitis. By postinoculation day (PID) 8, excessive bleeding and hematoma formation at venipuncture sites, dehydration, and diarrhea developed. At necropsy, the most consistent findings were oral lesions and widespread hemorrhage, which ranged from petechia to massive hematoma formation. Bluetongue virus caused progressive prolongation of activated partial thromboplastin time and prothrombin time, and progressive reduction of Factors VIII and XII plasma activities beginning on PID 6. A progressive decrease in platelet numbers also developed on PID 6. Changes in platelet size were not detected. Mean thrombin time was shortened, but prolongation developed in 1 deer. Mean fibrinogen concentration and Factor V plasma activity initially increased and then decreased, but remained above preinoculation values. Factor V activity was low in a few deer. Results of screening tests for inhibitors of the intrinsic coagulation system were positive in 2 deer. High concentrations of fibrin(ogen) degradation products were first detected between PID 3 and 6. Hematologic changes included leukopenia, lymphopenia, neutrophilia, and low total plasma protein concentration. Differences in PCV, hemoglobin concentration, or RBC counts were not detected between infected and control deer. Serum total bilirubin concentration increased by PID 6, primarily because of increased unconjugated bilirubin concentration. Mild to severe increases in serum aspartate transaminase activity were accompanied by more marked increases in creatine kinase activity. Indirect Coombs test results were negative in all deer.(ABSTRACT TRUNCATED AT 250 WORDS)

Bluetongue virus.

Bluetongue (BLU) is a noncontagious viral disease. The virus is a member of the Orbivirus genus and serves as the prototype virus of the genus. BLU is primarily a disease of domestic ruminants, some wild ruminants, and, recently, domestic dogs. The disease is caused by 1 of 24 different serotypes of virus that are distributed worldwide. This article reviews the viruses, their distribution, clinical signs, pathogenesis, and the roles they play in reproductive failure.

Using animal performance data to evidence the under-reporting of case herds during an epizootic: application to an outbreak of bluetongue in cattle.

Following the emergence of the Bluetongue virus serotype 8 (BTV-8) in France in 2006, a surveillance system (both passive and active) was implemented to detect and follow precociously the progression of the epizootic wave. This system did not allow a precise estimation of the extent of the epizootic. Infection by BTV-8 is associated with a decrease of fertility. The objective of this study was to evaluate whether a decrease in fertility can be used to evidence the under-reporting of cases during an epizootic and to quantify to what extent non-reported cases contribute to the total burden of the epizootic. The cow fertility in herds in the outbreak area (reported or not) was monitored around the date of clinical signs. A geostatistical interpolation method was used to estimate a date of clinical signs for non-reported herds. This interpolation was based on the spatiotemporal dynamic of confirmed case herds reported in 2007. Decreases in fertility were evidenced for both types of herds around the date of clinical signs. In non-reported herds, the decrease fertility was large (60% of the effect in reported herds), suggesting that some of these herds have been infected by the virus during 2007. Production losses in non-reported infected herds could thus contribute to an important part of the total burden of the epizootic. Overall, results indicate that performance data can be used to evidence the under-reporting during an epizootic. This approach could be generalized to pathogens that affect cattle's performance, including zoonotic agents such as Coxiella burnetii or Rift Valley fever virus.

Potential role of proinflammatory cytokines in the pathogenetic mechanisms of vascular lesions in goats naturally infected with bluetongue virus serotype 1.

In vitro studies have demonstrated that bluetongue virus (BTV)-induced vasoactive mediators could contribute to the endothelial cells dysfunction and increased vascular permeability responsible of lesions characteristic of bluetongue (BT) like oedema, haemorrhages and ischaemic necrosis in different tissues. However, few in vivo studies have been carried out to clarify the causes of these lesions. The aim of this study was to elucidate in vivo the pathogenetic mechanisms involved in the appearance of vascular lesions in different organs during BT. For this purpose, tissue samples from goats naturally infected with bluetongue virus serotype 1 (BTV-1) were taken for histopathological and immunohistochemical studies to determine the potential role of proinflammatory cytokines (tumour necrosis factor alpha, TNFα and interleukin one alpha, IL-1α) in the increased vascular permeability and their relationship with the presence of virus. Gross and histopathological examination revealed the presence of vascular damage leading to generalized oedema and haemorrhages. Immunohistochemical studies displayed that endothelial injury may have been due to the direct pathogenic effect of BTV infection on endothelial cells or may be a response to inflammatory mediators released by virus-infected endothelial cells and, possibly, other cell types such as monocytes/macrophages. These preliminary results of what appears to be the first in vivo study of tissue damage in small BT-infected ruminants suggest a direct link between the appearance of vascular changes and the presence of BTV-induced vasoactive cytokines.