Incidence of bovine neonatal pancytopenia in 243 farms in Germany

Background Several research groups from different European countries have worked on the aetiopathogenesis of bovine neonatal pancytopenia (BNP) and an association between the use of the vaccine PregSure BVD (Pfizer, Germany) and the development of this haemorrhagic disease was confirmed. Because BNP is not a notifiable disease, it is difficult to obtain information on its incidence. Based on pharmacovigilance (PhV) data, which are the only officially available data at the national level, the incidence of BNP is considered low. However, voluntary reporting of the disease can lead to underreporting. To gain more insight into the incidence of BNP among the affected herds, an epidemiological study was performed, which focused on 243 farms in Germany with cases of BNP. Farmers were asked to report the occurrence of BNP, including the number of cases, which allowed calculation of incidence in the affected herds. Matching such data with the registered cases in the National PhV System (NPhVS) gave us an opportunity to assess the extent of BNP underreporting. Results On 243 farms, a total of 1195 calves younger than 4 weeks with haemorrhagic diathesis were registered. In 58 % of the reports, a diagnosis of BNP was confirmed by blood analysis and or by necropsy. The number of cases observed on individual farms ranged from 1 to 80. Based on these results, the incidence of BNP on affected farms ranged from 0.3 to 15.2 % (median 2.9 %). The maximal incidence in the year with the highest number of BNP calves ranged between 0.4 and 18.6 % (median 3.3 %). Comparing the number of cases registered in the NPhVS to the numbers found in this study revealed considerable underreporting to the national database: only 44 % of the farms and 41 % of the BNP calves included in the study were registered in the NPhVS. Conclusions In spite of the opportunity to report BNP calves to the Paul-Ehrlich-Institut (Langen, Germany), the estimated number of undetected BNP cases is remarkably high. However, even if the revealed substantial underreporting is taken into account, the incidence of BNP is low. Nevertheless, the incidence on some affected farms is very high, resulting in considerable financial losses that should not be underestimated. Although the exact pathomechanism of BNP at the molecular level is still not known, its incidence is clearly declining following withdrawal of PregSure BVD from the market. Electronic supplementary material The online version of this article (doi:10.1186/s12917-016-0857-7) contains supplementary material, which is available to authorized users.


Background
First cases of bovine neonatal pancytopenia (BNP) were reported in Germany in 2006 and then in several other European countries and New Zealand [1]. The work of several research groups in various European countries has yielded important information about the aetiology and pathogenesis: The consumption of colostrum-containing alloreactive antibodies plays a crucial role in the development of BNP [2,3]. Vaccination against bovine viral diarrhoea virus (BVDV) was suspected to be correlated with the pathogenesis of BNP [4]. Intensive investigations identified the inactivated vaccine PregSure BVD (Pfizer, Germany) as a major risk factor for developing BNP [5][6][7]. In some cows, the PregSure BVD vaccine induces alloreactive antibodies that target antigens derived from bovine cells used for vaccine production [8]. Owing to the consumption of colostrum from these cows, some calves and foster calves develop thrombocytopenia, leukocytopenia, and panmyelophthisis [8][9][10]. As a consequence, affected calves show bleeding symptoms and most often, immunosuppression [2,11,12]. Owing to the absence of a promising therapy for BNP, only about 10 % of affected calves survive [4,10,11]. A subclinical course of the disease has been described [13]. The most important differential diagnoses for the clinical signs of BNP are the haemorrhagic form of BVD (type 2), a severe septicaemia, and other haemorrhagic diatheses. The haemorrhagic form of BVD is very uncommon in Germany; in 95 % of BVDV infections, the virus is type 1 [14]. Furthermore, bleeding disorders of cattle are relatively rare. If these disorders occur, they generally happen not only in calves within the first 4 weeks of age, but at all ages. No other haemorrhagic diatheses show similar processes to BNP [15]. Classic septicaemia affects newborn calves between 2 and 6 days of age, and is attributed to other diseases. Bleeding signs in septicaemic calves are possibly due to capillary fragility or thrombocytopenia, but they are less typical signs [16].
There is a remarkable difference in the incidence of BNP among different regions within a country [17]. In addition to this geographical difference, the number of cases per herd also varies [2,4,12]. BNP is not a notifiable disease and, therefore, exact figures about its occurrence are not available. Because the occurrence of BNP is believed to be related to the use of a vaccine, each case should be reported as a possible adverse event within the pharmacovigilance (PhV) scheme. Veterinary surgeons and farmers have the opportunity to report BNP cases to their national competent authority, which for Germany, is the Paul-Ehrlich-Institut in Langen. The agency recorded 4623 cases in Europe (date of download: 28.02.2011), including 3040 cases from Germany.. However, there is currently no information available as to what degree these numbers truly reflect the BNP incidence. Furthermore, little information exists about the incidence within an individual herd; most often, the information is based on investigations on single farms. According to reported results, within-herd incidence are no higher than 10 % [8].
The aim of our study was to provide an overview of the occurrence of BNP based on 243 affected farms in Germany. The incidence of BNP was calculated and the data of this study were compared with cases registered in the National PhV System (NPhVS) to identify to what extent BNP cases were reported within the PhV scheme.

Participating farms
Since the first case of BNP, affected calves have been registered in a database at the Clinic for Ruminants with Ambulatory and Herd Health Services, LMU Munich (Oberschleissheim, Germany). Calls to encourage registration from all over Germany were published in Deutsches Tieraerzteblatt, a journal of the German Veterinary Association. The database included 310 farms on January 3, 2011. Farms were eligible as case farms if they had one or more confirmed cases of BNP by necropsy (panmyelophthisis) and or by blood analysis (thrombocytopenia [less than 200 × 10 9 platelets (PLT)/L] and leukocytopenia [less than 4 × 10 9 white blood cells (WBC)/L]). Blood analyses were performed by the laboratory of the Clinic for Ruminants with Ambulatory and Herd Health Services, LMU Munich. Necropsies were performed by one of the following institutions: Bavarian Authority for Health and Food Safety in Oberschleißheim or Erlangen, Bavarian Animal Health Service in Poing, Institute of Veterinary Pathology, Center for Clinical veterinary Medicine, LMU Munich or Animal Health service in Fellbach, Baden-Wuerttemberg.
Twenty-seven farms were excluded, because the blood analysis of affected calves with haemorrhagic diathesis revealed only thrombocytopenia or leukocytopenia. Further, farms which only reported on BNP calves without any confirmed case, were excluded (n = 28). In total, 255 farms with at least one confirmed case of BNP were selected from the database. One year was defined as the 12 month period from the first of January until the 31st of December. During the interview, the answers were recorded on paper. In case that the herd manager needed more time to look up the information, the farm was contacted a second time. If the farmer did not have any information about the vaccine that was used to vaccinate against BVDV, the veterinarian was contacted. If the farm had changed the veterinarian during the considered timeframe, the former veterinarian was called to get the information needed. Only farms that used PregSure BVD were included in the analysis. The questionnaire is available as Additional file 1.

Incidence of BNP at the herd level
The total incidence on each farm was calculated as: The considered time period for each farm was not equal due to the fact that BNP occurred on some farms over several years and on other farms only within 1 year. The aim of the study was to evaluate the incidence of BNP on farms while the disease was prevalent on the farm. Using the calculation above, allowed the comparison of incidences across farms. In addition, the maximal incidence was calculated, which was related to the year with the highest number of calves with BNP on the farm:

Highest number of calves with BNP in one specific year Number calves born in that year
Â 100

Data matching between the NPhVS and the clinic for ruminants
Data matching was used to compare the number of farms and cases of BNP registered in the national PhV database at the Paul-Ehrlich-Institut against the numbers calculated in our study. Therefore, the first digits of the postal zip codes and the initials of the farmers were compared. Farms and cases not listed in the NPhVS were counted.

Descriptive and statistical analyses
The geographical distribution of the farms affected by BNP was illustrated in a map created in ArcGIS software (ESRI, version 9.3). A descriptive statistical analysis was conducted using Excel version 13.0 (Microsoft Inc., USA) and SPSS for Windows version 19 (SPSS Inc., USA). Binomial confidence intervals were calculated in Excel using the method of Agresti and Coull [18].

Participating farms
Two of the 255 contacted farmers were not willing to give an interview and one farmer had stopped farming, thus they were excluded from the analysis. Nine farms were excluded, because the herd was either not vaccinated against BVDV or PregSure BVD was not used.

Number of cases observed per farm
The number of newborn calves with haemorrhagic diathesis on each farm is illustrated in Fig. 3, excluding two farms with 50 and 80 cases, because they are outliers.
BNP cases were registered during only 1 year on nearly one-half of the farms (44 %). Thirty-five percent of the 241 farmers noted calves with BNP during two of the years, 36 farmers during three of the year, and another 12 farmers during four of the years. One interviewed owner reported cases during 5 years and another one during 6 years. Some farms had a year without having calves with BNP between the years with cases of BNP.
In 58 % of the registered cases, BNP was confirmed either by blood analysis and or by necropsy. In 42 % of the cases, farmers did not initiate an examination to clarify the aetiology of the bleeding disorder.

Overall incidence and incidence of BNP at the herd level
The incidence could not be calculated for two farms, because the farmers did not remember the exact distribution of BNP calves over the years. Regarding the The incidence of BNP within the 241 farms ranged between 0.3 and 15.2 % (median 2.9 %). The incidence distribution is shown in Fig. 4. The maximal incidence in the year with the highest number of BNP calves ranged from 0.4 to 18.6 % (median 3.3 %). The difference between the total incidence and the maximal incidence was particularly marked on farms with BNP cases scattered over several years, including 1 year with an irregularly high number of calves with BNP. On some of those farms, the maximal incidence was two to three times higher than the total incidence throughout the surveyed period.

Data matching between the NPhVS and the clinic for ruminants
Data matching between the two databases revealed 701 of 1195 cases (59 %) were not registered in the NPhVS, but were noted in the interviews conducted in our study. A total of 137 of the 243 participating farms (56 %) had not reported any cases to the NPhVS. Only 41 % of the interviewed Bavarian farmers and only 33 % of the 759 BNP calves from Bavaria in our study were registered with the NPhVS. Ten of 12 farms from Baden-Wuerttemberg reported their cases to the NPhVS,   Table 1.

Incidence of BNP at the herd level
One of the main objectives of this study was to identify the incidence of BNP at the herd level. For the calculation of the incidence the average number of calves born on the farm was available. This could have introduced a bias, as stillborn calves would not have been included in the denominator of the calculation. Using the average number of calves born on the farm also could have introduced a bias, which we cannot exclude. However, all farmers included in the study had reported that their herd size had been stable over the considered time period, therefore we are confident that the number of calves born over several years did not vary considerably. BNP affects calves during their first 4 weeks of life. Male Holstein-Frisian and Red Holstein calves are usually sold at an age of two weeks, thus it is possible that some cases of BNP were missed when BNP occurred after the calves were sold to another farm. However, most of the participating farms (77 %) had German Fleckvieh, crossbred or Brown Swiss. Male calves from those farms are typically not sold before they have reached a certain age or weight and do not leave the farm during the time when BNP may occur.
The highest incidence reported in a herd from our study (19 %) was substantially higher than the incidence rates reported in other studies, which was up to only 10 % [8]. Although the BNP incidence was low on most of the affected farms, some herds were obviously more severely affected. The incidence of BNP varies markedly among different countries and also among the federal states of Germany [4,6]. The farms enrolled in our study were from nine federal states of Germany. However, most of the farms with cases of BNP were localized in Fig. 4 Within-herd incidence of bovine neonatal pancytopenia (BNP). The incidence was calculated using the number of BNP calves on 241 affected farms from January 1, 2005, to March 31, 2011. The rate ranged from 0.3 to 15.2 % (median 2.9 %). The incidence could not be calculated for two farms, because the exact distribution of BNP cases over the years was not available Bavaria. Kasonta et al. [17] investigated the influence of different patterns of PregSure BVD vaccinations on the incidence of BNP. Their data showed that different vaccination regimens resulted in differences in alloreactive antibody concentrations. Therefore, the differences in incidence rates among federal states can potentially be explained by the different vaccination protocols that exist. Apart from the varying incidence among the federal states of Germany, we found considerable differences among the participating farms within the federal states. Presumably, the use of different vaccination regimens at the herd level is the reason for that observation, but other factors (e.g. colostrum management) could have had an influence on the incidence as well.
The present study shows a remarkable increase of BNP cases in 2008, reaching a peak in 2009. Although PregSure BVD was licensed in 2004, an increased BNP incidence was not noted until 2007. The same phenomenon was observed in New Zealand: the first cases of BNP occurred there in 2011, which was 3 years after the introduction of PregSure BVD [19]. Kasonta et al. [17] interpreted the temporal delay as being associated with the repeated immunizations that are required for creating a titre of alloreactive antibodies high enough to induce clinical BNP in the calves.
Various reasons exist for the decline of BNP cases on farms in our study since 2010. Owing to increased knowledge concerning aetiopathogenesis, farmers adapted their management practices, especially colostrum and vaccination management [20]. The safest way to prevent further BNP cases is to dispose of colostrum coming from known 'BNP dams' and to feed calves colostrum from cows that have not been vaccinated with PregSure BVD. Before PregSure BVD was withdrawn from the German market in April 2010, many farmers had already switched to a different BVD vaccine or discontinued vaccination against the BVDV. Nevertheless, calves with BNP are still observed on some farms, but in much smaller numbers [21]. Therefore, the antibody titre seems to remain high for a long time, even though PregSure BVD is no longer used. It is uncertain how much longer further cases of BNP will occur, but they will most likely stop when the last PregSure BVD-vaccinated cows are removed.

Data matching between the two databases
Another objective of our study was to investigate whether the number of calves with BNP registered at the NPhVS reflects the real number of cases in Germany. More than one-half of the cases and farms had not been listed in the NPhVS database of the Paul-Ehrlich-Institut. Three-hundred cases of BNP were reported in our study that occurred before 2009, which was the year when BNP was recognized as a potential serious adverse effect of PregSure BVD. A retrospective report of these cases was technically possible, but seems unlikely to happen, so most of these cases are most likely not in the database. Although veterinarians are requested to report adverse events in accordance with the veterinary medical association's professional code of conduct, they and the farmers do not directly benefit from reporting cases to the NPhVS. Thus, they are increasingly reluctant to fill in reporting questionnaires. Although we discovered a remarkable extent of underreporting, considering the population of 4.3 million dairy cows in Germany [22], the overall incidence is still very low.

Economic relevance
The severity of economic losses in affected herds due to medical treatments of BNP calves, costs for the veterinarian, and especially case fatalities, is difficult to estimate. However, an appreciable pecuniary loss can be assumed for farms with high incidence. In addition, less obvious losses could be caused by subclinical BNP cases in which calves could be more susceptible to other diseases, especially infections due to immunosuppression. Thus, in an epidemiological study, 2.5 % of control calves had thrombocytopenia and leucocytopenia without clinical signs of BNP [15], and another research group induced subclinical BNP in 20 % of calves after feeding with colostrum from 'BNP dams' [23].

Confirmation of BNP via blood analysis or necropsy
To reduce the bias of misclassification and, thus, maximize the probability of including farms with actual cases, only farms with at least one confirmed case of BNP were included in this study. Therefore, we excluded some farms from the study because even though the farmers had observed calves with signs consistent with BNP within their herds, they did not initiate an examination to verify the aetiology.
As reflected by the high proportion (58 %) of cases that were confirmed by haematology and or necropsy, veterinary practitioners demonstrated a great commitment to elucidate the previously unknown aetiology of this disease.
The spectacular clinical signs that are most often seen in affected calves, and the severe process of BNP are very similar among cases. Therefore, it can be assumed that affected farmers and their veterinarians are aware of the disease pattern and that most of the calves included in this study without disease confirmation likely did have BNP. Some cases of BNP were probably missed at the very beginning of the occurrence when farmers and veterinarians were not aware of the disease or in case of sudden death. Furthermore it is not possible to get any information about subclinical cases of BNP within a herd without blood sampling the calves, thus subclinical BNP could not be taken into account.
Because there are no advantages (e.g. compensation or specific treatments and better prognoses for the calves after disease diagnosis) for farmers and veterinarians in confirming additional cases, the proportion of investigated cases will decline and reports on the occurrence of BNP to official agencies will become less common over the years.

Conclusions
The number of calves with BNP in Germany is higher than officially assumed. Many cases have not been registered as PhV reports at the NPhVS, and we revealed a remarkable underreporting of the disease.
The incidence of BNP varies considerably, not only among federal states, but also among affected herds within one area. Considerable financial losses can be assumed, especially on farms with a high incidence.
Considering the whole bovine population of Germany, the incidence of BNP is low, even when accounting for the fact that many affected calves have not been registered.
Due to the well-advanced clarification of the aetiopathogenesis of BNP and the suspension of the license for PregSure BVD, the peak incidence for this disease has passed. However, a few cases of BNP still occur. It is likely that cases of BNP will occur until the last dam vaccinated with PregSure BVD has left the farms.