**4. Discussion**

Where of response to a single serovar, which occurred in 238 samples, serovar Hardjoprajitino was obtained in 67.4%. Pyrogenes in 41 (11.6%), Pomona in 29 (8.51%) and Wolffi in 18 (5.09%). Hardjo is the serovar most commonly found in cattle, the species considered its primary maintenance host [3]. Serologically identical but genetically distinct types of serovars Hardjo exist: *L. interrogans*, serovar Hardjo, type Hardjoprajitno and *L. borgpetersenii*, serovar Hardjo, type Hardjobovis [11]. The chief cattle infectant serovars are Hardjo, Pomona, Gripptyphosa, Icterohaemorrhagiae, Wolffi and Canicola [12]. These were found in the present study at different times with varied percentages both in G-1 and in G-2.

Results show that the serovars are practically the same, seroprevalence varying in both groups with G-2 displaying the greatest differences for most serovars. This shows the importance of environmental contamination and indirect transmission, mainly by water and food. According to Lenharo et al. [13], this serovar is commonly found in wild mammals and these can act as sources of soil contamination and animal infection.

Serovars Bratislava, Djasiman, Hebdomadis, Icterohaemorrhagiae, Pomona and Tarassovi are considered incidental in cattle and indirect transmission is associated with contact with an environment contaminated by leptospires mainly from wild species or other domestic species [8]. On the other hand, serovars Pomona, Grippotyphosa and Icterohaemorrhagiae are frequently identified in incidental infections in cattle and their transmission related to pigs, rodents and wild animals [4, 14]. Bovines can host incidental serovars for an uncertain period [15].

Serovar Hardjoprajitino is responsible for decreases in cattle milk production and conception rates. Also commonly found in pigs, Wolffi is antigenically similar to Hardjo and cause of reproductive disorders and abortions in wild animals and therefore a source of environmental contamination. Serovar Pyrogenes is frequently found in *Rattus norvegicus* and can be considered an incidental contaminant for cattle [6]. Infection by Hardjobovis is frequently observed in cattle in several countries in subclinical forms associated to abortion, while serovar Hardjoprajitno, found in some countries, is characterized as more pathogenic and leading to reductions in milk production and reproductive problems [16].

Serovars Hardjobovis and Hardjoprajitino are adapted to cattle and cause the reproductive and the sudden milk production decrease syndromes. The first is related to serovar Hardjobovis and is characterized by miscarriage, stillbirths, infertility and weak calves. The latter is due to serovar Hardjoprajitino, characterized by udder flaccidity and a sudden decrease in milk production lasting from 2 to 10 days with changes in its consistency and colostrum [17].

Where response was observed for two serovars, the predominance of Hardjoprajitino serovars in 96 of the samples (41.9%), Pyrogenes 53 samples (23.1%), Pomona 20 samples (8.73%), Hardjobovis 18 samples (7.8%), Wolffi 16 samples (6.78%) and Guaricura in 13 samples (5.67%) was noted. The serological response can be influenced by the cross-detection between serovars of the same serogroup. Serovars Pomona, Grippotyphosa and Icterohaemorrhagiae are frequently identified in incidental infections in cattle and their transmission is related to pigs, rodents and wild animals [4, 14].

### *Serological Monitoring for* Leptospira *Spp. and Monitoring of Productive and Reproductive… DOI: http://dx.doi.org/10.5772/intechopen.98983*

Predominance of Hardjoprajitino serovars with 25 samples (25.7%), Pyrogenes 21 (21.6%), Pomona 12 (12.37%), Wolffi 15 (15.4%), Copenhageni 9 (9.27%), Guaricura 6 (6.18%) and Hardjobovis 5 (5.15%) samples was noted. Cattle is considered maintenance host for serovars Hardjoprajitno and Hardjobovis which are transmitted by urine associated with reproductive failures [2, 7]. Cross-reactions occur between different serogroups, mainly in the acute phase of the disease [17, 18]. The serovar Icterohaemorrhagiae found in the present study falls within the One Health concept mainly due to the presence of rodents [19]. On the other hand, participation of serovar Pyrogenes among the serovars that stand out at different times is highlighted in G-1 for moments 4, 5 and 6. According to Lenharo et al. [20] this serovar is commonly found in wild mammals, which can contaminate the soil and can infect animals.

Pregnancy decreased at moments 3, 4, 5, 6 and 8 in G-2 and at moments 5 and 9 in group G-1. In a study with 25 dairy herds, totaling 500 cows, 32% of the herds were positive for the *Sejroe* serogroup. Of the 500 cows studied, 48 (9.6%) were sera reactive, 38 (7.6%) with 400 IU titers and 10 (2%) ≥ 800 IU. Estrus repetition was the most reported reproductive problem and strongly associated with leptospirosis [21]. Milk production decreased in G-2 at moments 4, 5, 6, 7 and 9 and in G-1 at moments 5 and 9.

Seroprevalence, milk production and pregnancy rate are influenced by environmental contamination from animal urine, particularly regarding serovar Hardjo. This serovar decreases fertility, while Hardjoprajitino is related to milk production, which is in line with the reduction in liters of milk at moment 6 [16]. Increased rainfall contributes to the spread of the agent in both groups. This is a relevant aspect to be considered in zoo-sanitary management in relation to bovine leptospirosis since the environment has an important role in the chain of transmission of the disease [13]. The triad is thus complete: animal, infectious agent and environment plus human involvement which characterizes the idea of One Health since the disease is common to humans and animals.

With regard to the animals in G-2 and the production of liters of milk, there is a decrease at moment 6, in February, moment 7 in March and moment 9 in May. The lower milk production in these months may be related to the greater environmental contamination by lepspiras and therefore a reduction in output, possibly influenced by serovar Hardjoprajitino.

Pregnancy rates at Mo 5 were 75.5% in G-1 and 76.9% in G-2. Although figures were close, G-2 saw a slight increase. Milk production decreased in both groups. Preganancy rates and milk production are probably related to infection by serovar Hardjoprajitino. Rainfall increased significantly in October, November, December, January and February possibly favoring cross-contamination between the two groups.

The dog is the natural host of serovar Canicola and the brown rat (*Rattus norvegicus*) of serovars Icterohemorrhagiae, Copenhageni and Pyrogenes. Serovar Pomona has pigs, cattle and possums as its natural hosts while Grippotyphosa is found in the kidneys of wild animals such as rats, hares, martens and hamsters [22]. These animals can be sources of infection for cattle [3].

Hardjoprajitino is the serovar prevalent in cattle and responsible for decreased milk production and pregnancy rates, a fact observed in the present study. Pomona and Wolffi are adapted to swine and bovine species but Wolffi is frequently found in pigs and can also cause abortion in the final third of gestation, birth of weak fetuses and decreased conception rates [19].

Serovars Hardjoprajitino and Hardjo are the ones most frequently found in cattle and may cause productive and reproductive disorders [17]. Pomona is most commonly found in swine, which is adapted, however, it can infect cattle. Pomona is most commonly found in swine, to which it is adapted, it may infect cattle. Serovar Pyrogenes is found in the *Rattus norvegicus* species, implying a potential for environmental contamination. Rodent control and site management measures like waste removal and swamp land drainage are biosafety measures for preventing the spread of this serovar. Despite low at the studied moments, the occurrence of serovar Icterohamorrhagiae should be noted and its adaptation to the rodent species stressed [5, 13].

In order to investigate the effects of rainfall on leptospira infection in cattle, 582 animals were selected and samples from 362 of these collected in the rainy season and from 220 in the dry season. In the rainy season, seropositivity to MAT was 43.6% (158/362) and in the dry season 31.8% (70/220). The Sejroe serogroup predominated (54.8%; n = 125/228), the Javanica serogroup (16.2%; n = 37/228), Icterohaemorrhagiae (7.5%; n = 17/228) and Tarassovi (7.0%; n = 16/228). Seropositivity for incidental serogroups was more frequent in the rainy season (50.0%) than in the dry season (34.3%; p ≤ 0.0001) [23], reinforcing the environmental aspects of leptospirosis maintenance in cattle herds.

Reproductive failures such as early embryonic loss and consequent estrus repetition are increasingly associated with leptospiral infection. Although these failures are frequently associated with several factors, two studies with cattle revealed a strong association of estrus repetition with seroreactivity for the serogroup *Sejroe* [12, 21]. Contrary to the results obtained, according to Faine et al. [17] Hardjoprajitino is associated with decreases in milk production.

In the present study a greater participation of the serovar Hardjoprajitino, serogroup Sejroe, was also observed however the correlation between milk production and pregnancy rates in both G-1 and G-2 had no statistical significance with p > 0.05. A limiting aspect is the impossibility of comparing the results of both the dynamics of antibodies and those of milk production and pregnancy rate, as in the present study, since no similar research with two groups of animals living under the same environmental and management conditions on the same property can be found in the literature.

Although in the present study there was no statistical association (p > 0.05) between milk production and seropositivity in both groups, except for the months of May and August, which may be associated with a drop in temperature, when results were analyzed for each groups separately, G-1 showed a decrease in pregnancy rate at moments 5, 6, 7, 8 and 9 and in milk production at moments 5, 6, 7, 8 and 9, related to January (Mo 5) 161.6 mm (Ciagro – Centro Integrado de Informações Agrometeorológicas) and February (Mo 6) 363.3 mm rainfall. Those were months of high rainfall favoring environmental contamination. In G-2 the pregnancy rate decreased at moments 2, 3, 4, 5, 6, 7 and 8, October (Mo 2) 234.4 mm, November (Mo 3) 135.2 mm, December (Mo 4) 137.8 mm, January (Mo 5) 161.6 mm and February (Mo 6) 363.3 mm, all months with high rainfall. Productivity decreased at moments 4, 5, 6, 7 and 9.

There was no statistical association between pregnancy rate and seropositivity, p > 0.05 in either group. There was also no statistical association (p > 0.05) between milk production and positivity in either group except in May and August, when there was a decrease in milk production which may be related to food management, temperature drop and health of the mammary gland as a result of probable cases of mastitis. The property carrying out somatic cell counting (SCC) of milk samples from the expansion tank but not from individual animals was a limiting factor.

According to Ellis [4], bovine leptospirosis is most often caused by strains adapted from the serogroup Sejroe. bovine leptospirosis is most often caused by

### *Serological Monitoring for* Leptospira *Spp. and Monitoring of Productive and Reproductive… DOI: http://dx.doi.org/10.5772/intechopen.98983*

strains adapted from serogroup *Sejroe*. In these cases, disease acute phase may be subclinical except for infections in lactating cows where agalactia may occur. Clinical cases are less frequent and can represent outbreaks, the disease then characterized by abortions at any time during pregnancy [24, 25], albeit more frequent in the average period of pregnancy [3].

Seropositivity for leptospira and clinical cases of leptospirosis are often associated with environmental risk factors, such as rain and floods [26]. For the *Sejroe* serogroup, specifically the Hardjo genotypes, adapted to cattle, direct animal-toanimal transmission is more common than indirect transmission from environmental contamination. On the other hand, infections by incidental serovars by serogroup Icterohaemorrhagiae or Pomona lead to renal excretion. Transmission in incidental infections is more dependent on the presence of other host species and environmental factors, especially accumulated water [4].

Research on leptospiral DNA in the vaginal secretion of apparently asymptomatic cows reinforces the belief that in addition to environmental contamination infection can occur from female to male through vaginal discharges and secretions during natural mating [27]. This can hamper control programs by maintaining infection and disease endemic in the property.

For the Copenhageni, Pomona, Wolffi and Prajitino serovars, frequency of positive titers greater than 800 IU was significant, with p < 0.05, in the comparison of positive reagent greater than negative reagent. Cattle infected with adapted strains, including those related to cases of agent isolation [28] the property, often have low antibody titers [10].

Although leptospires can be detected in the urine of cattle infected with adapted strains [10], leptospiruria is intermittent and not very intense [4, 29]. Serovars Pomona, Grippotyphosa and Icterohaemorrhagiae are frequently identified in incidental infections in cattle and their transmission is related to pigs, rodents and wild animals [4, 14].

Infection transmission by incidental serovars is more dependent on the presence of other host species and environmental factors. A high percentage of isolation of the serovar Hardjo from the genital tract of cows is emphasized, suggesting tropism for that region [16]. Also, according to Ellis [4], as previously mentioned, the genotypes of Hardjo serovars are adapted to cattle and associated with the chronic reproductive form of leptospirosis.

The farm where the present study was developed carries out vaccination against leptospirosis every four months and elevated titers such as 800 IU, 1600 IU and 3200 IU were found. In vaccinated cattle, post-vaccination IgM and IgG titers are low (between 100 and 400) and transient between four to six months after vaccination [3]. This fact reinforces the possibility of the higher titers having been produced in response to infection.

With regard to milk production and pregnancy rates, Ellis [16] demonstrated relationship with serovar Hardjoprajiino, a result also found in the present study which corroborates the findings of reductions in milk production and pregnancy rates at the moments when Hardjoprajiino was the most detected serovar. Comparative discussion regarding data from the literature in similar studies is hindered due to the scarcity of research on infection dynamics with different groups of animals. The present study showed that the several serovars are maintained in the two groups of animals (G-1) and (G-2), that seroprevalence is also variable, and that some serovars show greater importance in these groups. It can also be observed that milk production and pregnancy rates decreased at those moments when the frequency of a given serovar, like Hardjobovis, increased.
