The observed seroprevalence of bTB in brucellosis-vaccinated cows in this herd was above the maximum 26% reported data in Holstein herds with high bBR prevalence in the same zone where the current study took place (Mellado et al. 2014). Despite the existence of a program to control this disease in this large dairy basin, the value of RB51 whole-herd vaccination, in combination with test-and-slaughter in controlling bovine brucellosis, has not been demonstrated. However, it has been observed that some farmers are reluctant to get rid of seropositive cows to bBR since they do not perceive severe symptoms in their cows as causing drastic reduction in productivity (Mellado et al. 2021). These seropositive cows are latent carriers of B. abortus within the herd regardless of the generalized vaccination of all cows. Thus, this failure to adequately control animal brucellosis might explain the extremely high proportion of brucellosis vaccinated cows seropositive to bBR. It has been observed that in brucellosis endemic areas, under intermediate prevalence conditions, there are high percentages of reactors, as well as abortions.
The interval between vaccination and the assay was very close in some cows, but cattle vaccinated with strain RB51 fail to produce antibodies that can be detected by conventional serologic tests (Stevens et al. 1995; Tittarelli et al. 2008). Thus, it is not expected that RB51 induces antibodies against smooth lipopolysaccharide (LPS) detectable by the serological tests used.
Older heifers at first calving in the current study were less likely to be seropositive to bBR than younger heifers at first calving. This response may be associated with an increased risk of dystocia in lighter heifers (Atashi et al. 2021) and the sequelae of this reproductive disorder, such as digestive and respiratory disorders, and retained placenta, uterine diseases and mastitis (Lombard et al. 2007). This physiological imbalance alters the immune function (Ingvartsen and Moyes 2013), which may predispose younger heifers to result seropositive to bBR. It is also likely that heifers calving at younger ages are more likely to be positive to bBR because they partition greater energy towards growth during their first lactation than heifers older at their first calving (Van Amburgh et al. 1998).
One of the most surprising findings in the present study was that neonatal diarrhea was associated with higher odds for seroprevalence to bBR. The opposite would be suspected, because preweaning diarrhea is a major factor for increasing the age of heifers for attaining puberty (Heinrichs and Heinrichs 2013), and older heifers at first calving were less susceptible to be seropositive to bBR in the current study. However, it is unclear how preweaing diarrhea could be associated with seropositivity to bBR. Its effect could be indirect via the occurrence of other diseases linked to diarrhea, such as bovine respiratory disease (Pardon et al. 2013), and these diseases may impair the immune function that could predispose heifers to acquire bBR.
An important finding of this study was that retained fetal membranes were significantly associated with seropositivity to bBR. This finding is consistent with Merga Sima et al. (2021). This association could be due to the immunosuppression experienced by the dairy cows during the transition period, which represents a time of physiological stress that results in defective separation of fetal membranes and, consequently, their retention post-calving. In fact, an important pathogenic factor causing retained fetal membranes is immune alteration during parturition (Mordak and Anthony 2015; Dervishi et al. 2016; Lu et al. 2020), although other reports indicate that cows with or without retained placenta showed similar immune function (Yazlık et al. 2019). Thus, it could be that disruption of the immune response in brucellosis-vaccinated cows with retained placenta may predispose these animals to result seropositive to bBR.
The odds of seropositivity to bBR in the group of heifers receiving two doses of the brucellosis vaccine were markedly greater than that of heifers that were vaccinated once. It has been stated that RB51 is an attenuated rough strain vaccine that, after vaccination, gives no false positives with the conventional serological assays. Thus, this vaccine is not supposed to interfere in brucellosis surveillance (Herrera-Lopez et al. 2010). However, in a field trial, 49% of pregnant cows from a brucellosis-free herd, revaccinated with the RB51vaccine, and introduced into an infected herd were seropositive to bBR using the card test (Leal-Hernandez et al. 2005). These data tear down the notion that RB51 vaccination does not induce antibodies that interfere with Brucellosis diagnosis.
After a first vaccination with strains RB51, a second inoculation induce a strong and complex immune response (Dorneles et al. 2015; Boggiatto et al. 2019). Furthermore, the presence of seropositive cows post-vaccination represents a secondary response that does not necessarily indicate brucellosis infection when confirmed by the Rivanol test (Cantú et al. 2007). Thus, in the current study, revaccination with the RB51 vaccine could have created persistent titres that confounded the identification of infected heifers by the card tests.
Heifers calving in summer were more likely to become seropositive to bBR than heifers calving during all other seasons, suggesting that heat stress in the lactating dairy heifers is positively associated with seropositivity to bBR. This hypothesis is proposed due to cows' immunosuppression due to heat stress (temperature-humidity index in summer > 88 units in the study site; Bagath et al. 2019; Lendez et al. 2021). Additionally, hot environments facilitate infectious microorganisms shedding (Hamel et al. 2021). Indirectly, hot weather can influence seropositivity to bBR mediated through reduced dry matter intake, and the negative energy balance (Wheelock et al. 2010) impairs immune function (Ingvartsen and Moyes 2013).
The risk of abortion was more likely in heifers seropositive to bBR than in seronegative animals. This response agrees with other studies, where positivity to brucellosis constitutes a major factor for abortion in cattle (Kardjadj 2018; Sarangi et al. 2021). However, even in seronegative heifers, a high percentage of abortions occurred, suggesting that vaccination with RB51 did not completely prevent abortion in heifers. These results agree with Poester et al. (2006) data, who observed that vaccination with RB51 partially prevented abortions in crossbreed virgin heifers.
Heifers with a BCS ≥ 3. 5 units at parturition had a much lower risk of abortion than heifers with BCS < 3.5 units. These results are in line with observations of Starbuck et al. (2004), who observed that cows in average body condition (2.75–3.25) sustained 92.1% of pregnancies, whereas those with BCS ≤ 2.50 maintained 84.2% of pregnancies. Likewise, Mellado et al. (2019) reported 10 percentage points higher fetal losses in cows with BCS < 3.5 than cows with higher BCS. Nutritional status influences the establishment and maintenance of a pregnancy to term in dairy cows (Meikle et al. 2018). BCS around 3.5 minimize BCS-related health and fertility disorders (O’Hara et al. 2015), which would explain the lower likelihood of abortion in heifers with greater body energy reserves at calving. Additionally, heifers with lower BCS tend to eat more, which increases progesterone clearance, resulting in decreased blood concentration of this hormone (Reksen et al. 2002), which is essential for pregnancy maintenance. Also, reduced BCS has long-term carryover detrimental effects on embryo quality (Carvalho et al. 2014). It is worth mentioning that no interactions were observed between various heifers-related variables and abortion, thus, suggesting an independent effect of BCS on the occurrence of this reproductive disorder.
In agreement with the conclusions of Mellado et al. (2019), in high-yielding Holstein cows, retained fetal membranes increased the risk of abortion in the current study. This response is not clear because current therapies to treat retained placenta effectively prevent a reduction of the reproductive performance in cows suffering from this reproductive disorder (Mellado et al. 2018). Therefore, it could be a carryover effect of uterine diseases (retained placenta and metritis) on the occurrence of abortion. These carryover effects might last longer than 4 mo and involve reduced oocyte competence and impaired uterine environment due to trauma to the endometrium (Ribeiro et al. 2016; Sheldon and Owens 2017).
The current study presents compelling evidence that heifers suffering from pneumonia during the growing period had an increased risk of abortion. This association is challenging to interpret and explain. It could be that pneumonia could have a greater risk for long-term sequelae as it happens in humans (Grimwood and Chang 2015), or occasionally may have a long-term persistence (Hermeyer et al. 2012), which could cause problems for unborn calves. Furthermore, with the occurrence of clinical diseases (calving problem, metritis, clinical endometritis, mastitis, pneumonia, digestive problems, and lameness), Ribeiro et al. (2011) documented the increased pregnancy loss, which suggests that these diseases have a profound impact on the maintenance of gestation.
Clinical metritis had a marked impact on the occurrence of abortion in heifers, which suggests that uterine infection is associated with fetal losses in dairy cows, even after the resolution of infection. These results align with Figueiredo et al. (2021), who reported that pregnancy loss tended to be greater for cows diagnosed with metritis but with failure of clinical cure following antimicrobial therapy. Likewise, Giuliodori et al. (2019) documented a greater likelihood of late embryonic loss in dairy cows diagnosed with clinical metritis. However, the mechanisms responsible for this persistent infertility are unclear and complicated to elucidate because metritis is associated with other peripartum disorders and metabolic challenges associated with the onset of lactation. This response could be explained by the potentially greater endometrial injury or incomplete uterine recovery in heifers undergoing clinical metritis. Also, uterine infections may reduce oocyte quality (Piersanti et al. 2020) and its capacity to develop to morulae (Dickson et al. 2020), which could have a carryover effect that could interfere with the completion of fetal development. An additional possible scenario is that the impact of puerperal metritis on the immune system persists weeks after the uterine inflammation has been resolved (Magata et al. 2016); therefore, there are long-term alterations of systemic immune responses which possibly interfere with the maintenance of pregnancy.